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<infon key="license">CC BY</infon>
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<infon key="article-id_doi">10.1038/srep23641</infon>
<infon key="article-id_pii">srep23641</infon>
<infon key="article-id_pmc">4806292</infon>
<infon key="article-id_pmid">27009356</infon>
<infon key="elocation-id">23641</infon>
<infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
<infon key="name_0">surname:Klima;given-names:Martin</infon>
<infon key="name_1">surname:Tóth;given-names:Dániel J.</infon>
<infon key="name_10">surname:Humpolickova;given-names:Jana</infon>
<infon key="name_11">surname:Nencka;given-names:Radim</infon>
<infon key="name_12">surname:Veverka;given-names:Vaclav</infon>
<infon key="name_13">surname:Balla;given-names:Tamas</infon>
<infon key="name_14">surname:Boura;given-names:Evzen</infon>
<infon key="name_2">surname:Hexnerova;given-names:Rozalie</infon>
<infon key="name_3">surname:Baumlova;given-names:Adriana</infon>
<infon key="name_4">surname:Chalupska;given-names:Dominika</infon>
<infon key="name_5">surname:Tykvart;given-names:Jan</infon>
<infon key="name_6">surname:Rezabkova;given-names:Lenka</infon>
<infon key="name_7">surname:Sengupta;given-names:Nivedita</infon>
<infon key="name_8">surname:Man;given-names:Petr</infon>
<infon key="name_9">surname:Dubankova;given-names:Anna</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">6</infon>
<infon key="year">2016</infon>
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<text>Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein</text>
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<text>in vitro reconstitution</text>
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<text>PI4KB</text>
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<text>ACBD3</text>
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<infon key="section_type">ABSTRACT</infon>
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<text>Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.</text>
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<text>Golgi adaptor protein</text>
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<text>acyl-coenzyme A binding domain containing protein 3</text>
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<text>PI4KB</text>
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<text>ACBD3</text>
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<text>enzymatic activity</text>
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<text>ACBD3:PI4KB</text>
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<infon key="section_type">INTRO</infon>
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<offset>1035</offset>
<text>Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals.</text>
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<text>Phosphatidylinositol 4-kinase beta</text>
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<text>PI4KB</text>
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<text>PI4K IIIβ</text>
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<text>human</text>
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<text>phosphatidylinositol</text>
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<text>phosphatidylinositol 4-phosphate</text>
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<infon key="section_type">INTRO</infon>
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<text>PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.</text>
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<infon key="section_type">INTRO</infon>
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<text>Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.</text>
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<text>Acyl-coenzyme A binding domain containing protein 3</text>
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<text>ACBD3</text>
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<text>giantin</text>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T14:01:06Z</infon>
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<infon key="updated_at">2023-09-21T14:01:32Z</infon>
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<infon key="updated_at">2023-09-21T14:01:16Z</infon>
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<text>metal transporter</text>
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<infon key="updated_at">2023-09-21T14:01:19Z</infon>
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<text>DMT1</text>
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<infon key="annotator">cleaner0</infon>
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<text>monomeric</text>
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<infon key="updated_at">2023-09-21T14:01:24Z</infon>
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<infon key="updated_at">2023-09-21T14:01:28Z</infon>
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<infon key="updated_at">2023-09-21T14:01:43Z</infon>
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<text>iron</text>
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<infon key="updated_at">2023-09-21T14:01:37Z</infon>
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<infon key="updated_at">2023-09-21T13:55:13Z</infon>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:02:16Z</infon>
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<text>polyglutamine repeat</text>
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<infon key="score">0.9988851</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:38:02Z</infon>
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<text>mutant</text>
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<infon key="updated_at">2023-09-21T14:01:55Z</infon>
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<text>huntingtin</text>
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<infon key="updated_at">2023-09-21T14:01:50Z</infon>
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<annotation id="80">
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<infon key="updated_at">2023-09-21T14:01:24Z</infon>
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<infon key="updated_at">2023-09-21T14:02:24Z</infon>
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<text>Rhes</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:02:29Z</infon>
<infon key="identifier">PR:</infon>
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<text>Dexras2</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<text>ACBD3</text>
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<infon key="score">0.9963929</infon>
<infon key="type">taxonomy_domain</infon>
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<infon key="updated_at">2023-09-21T13:57:20Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:02:53Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:03:03Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>picornaviruses</text>
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<infon key="updated_at">2023-09-21T14:03:21Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>poliovirus</text>
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<infon key="updated_at">2023-09-21T14:03:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3716" length="17"/>
<text>coxsackievirus B3</text>
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<annotation id="88">
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3754</offset>
<text>We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.</text>
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<infon key="updated_at">2023-09-21T14:03:42Z</infon>
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<text>biochemical and structural characterization</text>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
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<text>ACBD3</text>
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<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:10Z</infon>
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<text>enzymatic activity</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<location offset="4091" length="5"/>
<text>PI4KB</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="4097" length="6"/>
<text>kinase</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>4156</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>4164</offset>
<text>ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4164" length="5"/>
<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>4240</offset>
<text>In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T14:05:39Z</infon>
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<infon key="updated_at">2023-09-21T14:05:34Z</infon>
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<text>deletion</text>
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<text>kinase</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<infon key="updated_at">2023-09-21T14:05:01Z</infon>
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<text>in vitro binding assay</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4628" length="5"/>
<text>ACBD3</text>
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<annotation id="644">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:30Z</infon>
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<text>co-purified with the NiNTA-immobilized</text>
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<annotation id="108">
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<infon key="updated_at">2023-09-21T14:05:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4684" length="14"/>
<text>His6GB1-tagged</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4699" length="5"/>
<text>PI4KB</text>
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<annotation id="110">
<infon key="score">0.9989556</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4769" length="26"/>
<text>mammalian two-hybrid assay</text>
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<annotation id="738">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:06:50Z</infon>
<location offset="4821" length="9"/>
<text>localized</text>
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<annotation id="111">
<infon key="score">0.96972895</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4855" length="8"/>
<text>Q domain</text>
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<annotation id="112">
<infon key="score">0.99847716</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4867" length="5"/>
<text>ACBD3</text>
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<annotation id="113">
<infon key="score">0.99634355</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4913" length="9"/>
<text>glutamine</text>
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<annotation id="674">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="4941" length="17"/>
<text>N-terminal region</text>
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<annotation id="114">
<infon key="score">0.99838805</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4962" length="5"/>
<text>PI4KB</text>
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<annotation id="115">
<infon key="score">0.9992541</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4982" length="14"/>
<text>helical domain</text>
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<annotation id="116">
<infon key="score">0.9902597</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5001" length="9"/>
<text>expressed</text>
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<annotation id="117">
<infon key="score">0.8818047</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5015" length="8"/>
<text>Q domain</text>
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<annotation id="118">
<infon key="score">0.9983418</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5027" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="119">
<infon key="score">0.99775714</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5043" length="7"/>
<text>241–308</text>
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<annotation id="675">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="5060" length="17"/>
<text>N-terminal region</text>
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<annotation id="120">
<infon key="score">0.99809617</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5081" length="5"/>
<text>PI4KB</text>
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<annotation id="121">
<infon key="score">0.9972978</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5097" length="4"/>
<text>1–68</text>
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<annotation id="122">
<infon key="score">0.9982603</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5106" length="7"/>
<text>E. coli</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>5275</offset>
<text>Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.</text>
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<infon key="score">0.99530846</infon>
<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5309" length="5"/>
<text>ACBD3</text>
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<annotation id="124">
<infon key="score">0.9589814</infon>
<infon key="type">oligomeric_state</infon>
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<infon key="updated_at">2023-09-21T14:06:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5319" length="8"/>
<text>dimerize</text>
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<infon key="updated_at">2023-09-21T14:05:05Z</infon>
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<location offset="5333" length="26"/>
<text>mammalian two-hybrid assay</text>
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<infon key="score">0.9991631</infon>
<infon key="type">complex_assembly</infon>
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<infon key="updated_at">2023-09-21T13:56:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5420" length="11"/>
<text>ACBD3:PI4KB</text>
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<infon key="score">0.9983687</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5453" length="26"/>
<text>sedimentation coefficients</text>
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<infon key="score">0.9965487</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5483" length="5"/>
<text>ACBD3</text>
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<infon key="score">0.9935821</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5493" length="5"/>
<text>PI4KB</text>
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<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:41Z</infon>
<location offset="5499" length="5"/>
<text>alone</text>
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<infon key="score">0.999072</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5509" length="11"/>
<text>ACBD3:PI4KB</text>
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<annotation id="131">
<infon key="score">0.9988844</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5548" length="30"/>
<text>analytical ultracentrifugation</text>
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<infon key="updated_at">2023-09-21T14:08:09Z</infon>
<location offset="5647" length="17"/>
<text>molecular weights</text>
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<infon key="score">0.99865687</infon>
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<infon key="updated_at">2023-09-21T14:01:50Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>monomeric</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:07:34Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5808" length="12"/>
<text>ACBD3: PI4KB</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="5920" length="8"/>
<text>Q domain</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5932" length="5"/>
<text>ACBD3</text>
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<location offset="5946" length="17"/>
<text>N-terminal region</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<location offset="5967" length="5"/>
<text>PI4KB</text>
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<infon key="score">0.9987138</infon>
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<infon key="updated_at">2023-09-21T14:07:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6068" length="11"/>
<text>full length</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6080" length="5"/>
<text>ACBD3</text>
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<infon key="score">0.99603987</infon>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6090" length="5"/>
<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T14:07:05Z</infon>
<infon key="identifier">MESH:</infon>
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<text>surface plasmon resonance</text>
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<infon key="updated_at">2023-09-21T14:07:09Z</infon>
<infon key="identifier">MESH:</infon>
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<text>SPR</text>
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<infon key="updated_at">2023-09-21T14:07:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6135" length="3"/>
<text>SPR</text>
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<infon key="updated_at">2023-09-21T14:07:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6189" length="2"/>
<text>Kd</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<location offset="6256" length="5"/>
<text>ACBD3</text>
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<infon key="score">0.99455845</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6266" length="5"/>
<text>PI4KB</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="6302" length="8"/>
<text>Q domain</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6314" length="5"/>
<text>ACBD3</text>
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<annotation id="676">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="6328" length="17"/>
<text>N-terminal region</text>
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<annotation id="146">
<infon key="score">0.99657923</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6349" length="5"/>
<text>PI4KB</text>
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<annotation id="147">
<infon key="score">0.9985013</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6384" length="21"/>
<text>dissociation constant</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>6446</offset>
<text>Structural analysis of the ACBD3:PI4KB complex</text>
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<infon key="score">0.9987158</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:09:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6446" length="19"/>
<text>Structural analysis</text>
</annotation>
<annotation id="149">
<infon key="score">0.9992986</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="6473" length="11"/>
<text>ACBD3:PI4KB</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>6493</offset>
<text>Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.</text>
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<infon key="score">0.9989967</infon>
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<infon key="updated_at">2023-09-21T14:07:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6493" length="11"/>
<text>Full length</text>
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<infon key="score">0.9978492</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6505" length="5"/>
<text>ACBD3</text>
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<annotation id="152">
<infon key="score">0.9978822</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6515" length="5"/>
<text>PI4KB</text>
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<annotation id="682">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:09:37Z</infon>
<location offset="6540" length="32"/>
<text>intrinsically disordered regions</text>
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<annotation id="153">
<infon key="score">0.9989658</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:09:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6610" length="45"/>
<text>hydrogen-deuterium exchange mass spectrometry</text>
</annotation>
<annotation id="154">
<infon key="score">0.9989092</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:09:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6657" length="6"/>
<text>HDX-MS</text>
</annotation>
<annotation id="683">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:10:09Z</infon>
<location offset="6733" length="11"/>
<text>well folded</text>
</annotation>
<annotation id="155">
<infon key="score">0.9973455</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:10:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6792" length="8"/>
<text>crystals</text>
</annotation>
<annotation id="684">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:10:31Z</infon>
<location offset="6831" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="156">
<infon key="score">0.99859995</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6875" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="647">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="6881" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="677">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="6898" length="17"/>
<text>N-terminal region</text>
</annotation>
<annotation id="157">
<infon key="score">0.9982198</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6919" length="5"/>
<text>PI4KB</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>6926</offset>
<text>For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1.</text>
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<infon key="score">0.8772636</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:10:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6958" length="20"/>
<text>isotopically labeled</text>
</annotation>
<annotation id="159">
<infon key="score">0.9981755</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6979" length="5"/>
<text>ACBD3</text>
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<annotation id="648">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="6985" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="160">
<infon key="score">0.9734254</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:10:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6998" length="20"/>
<text>isotopically labeled</text>
</annotation>
<annotation id="161">
<infon key="score">0.9956441</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7019" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="649">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="7025" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="162">
<infon key="score">0.9879413</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7034" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="678">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="7040" length="17"/>
<text>N-terminal region</text>
</annotation>
<annotation id="685">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:28Z</infon>
<location offset="7083" length="16"/>
<text>NMR spectroscopy</text>
</annotation>
<annotation id="679">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<location offset="7140" length="17"/>
<text>N-terminal region</text>
</annotation>
<annotation id="163">
<infon key="score">0.9971308</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7190" length="13"/>
<text>co-expression</text>
</annotation>
<annotation id="164">
<infon key="score">0.99493366</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7285" length="3"/>
<text>15N</text>
</annotation>
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<infon key="score">0.9964359</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7289" length="3"/>
<text>13C</text>
</annotation>
<annotation id="688">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:45Z</infon>
<location offset="7293" length="8"/>
<text>labelled</text>
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<infon key="score">0.9989409</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7381" length="4"/>
<text>free</text>
</annotation>
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<infon key="score">0.9986345</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7386" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="650">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="7392" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="690">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:09Z</infon>
<location offset="7440" length="14"/>
<text>2D 15N/1H HSQC</text>
</annotation>
<annotation id="168">
<infon key="score">0.99490064</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7455" length="7"/>
<text>spectra</text>
</annotation>
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<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:53Z</infon>
<location offset="7528" length="28"/>
<text>triple-resonance experiments</text>
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<infon key="score">0.99697316</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7607" length="3"/>
<text>15N</text>
</annotation>
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<infon key="score">0.97899354</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7615" length="2"/>
<text>1H</text>
</annotation>
<annotation id="171">
<infon key="score">0.9988808</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7627" length="4"/>
<text>free</text>
</annotation>
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<infon key="score">0.9985446</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7632" length="5"/>
<text>ACBD3</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="7638" length="8"/>
<text>Q domain</text>
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<infon key="type">residue_range</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:13:11Z</infon>
<location offset="7726" length="9"/>
<text>Met1-Lys4</text>
</annotation>
<annotation id="173">
<infon key="score">0.9995875</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:13:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7741" length="5"/>
<text>Gln44</text>
</annotation>
<annotation id="174">
<infon key="score">0.99882644</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7818" length="4"/>
<text>free</text>
</annotation>
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<infon key="score">0.9986002</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7823" length="5"/>
<text>ACBD3</text>
</annotation>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="7829" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="176">
<infon key="score">0.9974669</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7924" length="3"/>
<text>Gln</text>
</annotation>
<annotation id="177">
<infon key="score">0.99632865</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7935" length="3"/>
<text>Gln</text>
</annotation>
<annotation id="178">
<infon key="score">0.9996074</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:13:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7951" length="5"/>
<text>Gln44</text>
</annotation>
<annotation id="179">
<infon key="score">0.9996132</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:14:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7972" length="5"/>
<text>Gln48</text>
</annotation>
<annotation id="180">
<infon key="score">0.9880802</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:13:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8073" length="9"/>
<text>glutamine</text>
</annotation>
<annotation id="181">
<infon key="score">0.907675</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8159" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="182">
<infon key="score">0.99920267</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8261" length="3"/>
<text>Gln</text>
</annotation>
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<infon key="score">0.9972893</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8266" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="184">
<infon key="score">0.99957603</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:14:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8277" length="4"/>
<text>Ala2</text>
</annotation>
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<infon key="score">0.9949039</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8283" length="5"/>
<text>PI4KB</text>
</annotation>
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<infon key="score">0.907263</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:01Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>15N</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8406" length="3"/>
<text>13C</text>
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<annotation id="687">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:12:10Z</infon>
<location offset="8414" length="2"/>
<text>1H</text>
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<annotation id="188">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:16:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8475" length="4"/>
<text>NOEs</text>
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<annotation id="691">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:21Z</infon>
<location offset="8498" length="20"/>
<text>3D 15N/1H NOESY-HSQC</text>
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<annotation id="189">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:15:58Z</infon>
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<location offset="8523" length="17"/>
<text>13C/1H HMQC-NOESY</text>
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<annotation id="190">
<infon key="score">0.98942995</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:11:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8541" length="7"/>
<text>spectra</text>
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<annotation id="693">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:16:25Z</infon>
<location offset="8580" length="22"/>
<text>structural calculation</text>
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<annotation id="191">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:16:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8604" length="21"/>
<text>Structural statistics</text>
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<annotation id="192">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:16:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8662" length="10"/>
<text>structures</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>8698</offset>
<text>This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).</text>
<annotation id="193">
<infon key="score">0.998018</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8703" length="9"/>
<text>structure</text>
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<annotation id="194">
<infon key="score">0.99866307</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8731" length="8"/>
<text>Q domain</text>
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<annotation id="195">
<infon key="score">0.9987442</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8748" length="17"/>
<text>two helix hairpin</text>
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<annotation id="694">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:28Z</infon>
<location offset="8777" length="5"/>
<text>helix</text>
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<annotation id="695">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:28Z</infon>
<location offset="8813" length="5"/>
<text>helix</text>
</annotation>
<annotation id="196">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8851" length="18"/>
<text>three helix bundle</text>
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<annotation id="197">
<infon key="score">0.972546</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8900" length="5"/>
<text>helix</text>
</annotation>
<annotation id="198">
<infon key="score">0.99828124</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8913" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="199">
<infon key="score">0.9977347</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8940" length="5"/>
<text>44–64</text>
</annotation>
<annotation id="200">
<infon key="score">0.9990201</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8985" length="12"/>
<text>kinase helix</text>
</annotation>
<annotation id="201">
<infon key="score">0.9991654</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9024" length="12"/>
<text>kinase helix</text>
</annotation>
<annotation id="202">
<infon key="score">0.99960405</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9065" length="5"/>
<text>Val42</text>
</annotation>
<annotation id="203">
<infon key="score">0.9996008</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9072" length="5"/>
<text>Ile43</text>
</annotation>
<annotation id="204">
<infon key="score">0.99959177</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9083" length="5"/>
<text>Asp44</text>
</annotation>
<annotation id="205">
<infon key="score">0.9983835</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9167" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="206">
<infon key="score">0.99630404</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:13Z</infon>
<infon key="identifier">GO:</infon>
<location offset="9238" length="11"/>
<text>PI4KB:ACBD3</text>
</annotation>
<annotation id="696">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:37Z</infon>
<location offset="9250" length="28"/>
<text>interactions are hydrophobic</text>
</annotation>
<annotation id="207">
<infon key="score">0.9969462</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9301" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="208">
<infon key="score">0.9955851</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9343" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="209">
<infon key="score">0.99958163</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9349" length="6"/>
<text>Tyr261</text>
</annotation>
<annotation id="210">
<infon key="score">0.994023</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9360" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="211">
<infon key="score">0.9995528</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9366" length="5"/>
<text>His63</text>
</annotation>
<annotation id="212">
<infon key="score">0.9955701</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9402" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="213">
<infon key="score">0.99957126</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:27:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9408" length="6"/>
<text>Tyr288</text>
</annotation>
<annotation id="214">
<infon key="score">0.9939761</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9423" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="215">
<infon key="score">0.99950206</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:26:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9439" length="5"/>
<text>Asp44</text>
</annotation>
<annotation id="216">
<infon key="score">0.99854785</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9490" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="217">
<infon key="score">0.9942094</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9496" length="5"/>
<text>helix</text>
</annotation>
<annotation id="218">
<infon key="score">0.99885774</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9505" length="11"/>
<text>amphipathic</text>
</annotation>
<annotation id="219">
<infon key="score">0.991676</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9525" length="19"/>
<text>hydrophobic surface</text>
</annotation>
<annotation id="220">
<infon key="score">0.9984603</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9558" length="8"/>
<text>Q domain</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9580</offset>
<text>To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).</text>
<annotation id="221">
<infon key="score">0.8155627</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9599" length="15"/>
<text>structural data</text>
</annotation>
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<infon key="score">0.74476194</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9619" length="10"/>
<text>introduced</text>
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<infon key="score">0.96569216</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9642" length="15"/>
<text>point mutations</text>
</annotation>
<annotation id="224">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9715" length="24"/>
<text>in vitro pull-down assay</text>
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<annotation id="225">
<infon key="score">0.99917686</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9751" length="9"/>
<text>Wild type</text>
</annotation>
<annotation id="226">
<infon key="score">0.9988563</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9761" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="227">
<infon key="score">0.99860805</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:15:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9775" length="11"/>
<text>co-purified</text>
</annotation>
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<infon key="score">0.88533324</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9823" length="11"/>
<text>His6-tagged</text>
</annotation>
<annotation id="229">
<infon key="score">0.9991353</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9835" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="230">
<infon key="score">0.99864405</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9845" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="231">
<infon key="score">0.567764</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9871" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="232">
<infon key="score">0.99877995</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:29:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9877" length="4"/>
<text>V42A</text>
</annotation>
<annotation id="233">
<infon key="score">0.9988329</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:29:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9886" length="4"/>
<text>V47A</text>
</annotation>
<annotation id="234">
<infon key="score">0.9898858</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9891" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="235">
<infon key="score">0.8356905</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9913" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="236">
<infon key="score">0.9989662</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9941" length="17"/>
<text>binding interface</text>
</annotation>
<annotation id="237">
<infon key="score">0.99909496</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9960" length="4"/>
<text>I43A</text>
</annotation>
<annotation id="238">
<infon key="score">0.9991035</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9966" length="4"/>
<text>V55A</text>
</annotation>
<annotation id="239">
<infon key="score">0.9991026</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:29:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9972" length="4"/>
<text>L56A</text>
</annotation>
<annotation id="240">
<infon key="score">0.9991299</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:28:33Z</infon>
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<text>wild type</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:29:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10035" length="5"/>
<text>Y266A</text>
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:38:02Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>mutant</text>
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<infon key="updated_at">2023-09-21T14:29:18Z</infon>
<infon key="identifier">MESH:</infon>
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<text>mutant</text>
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<infon key="updated_at">2023-09-21T14:29:22Z</infon>
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<infon key="updated_at">2023-09-21T14:29:26Z</infon>
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<text>H284A</text>
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<infon key="updated_at">2023-09-21T14:29:30Z</infon>
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<text>Y288A</text>
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<infon key="updated_at">2023-09-21T14:28:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10125" length="7"/>
<text>mutants</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
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<text>ACBD3 efficiently recruits the PI4KB enzyme to membranes</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<location offset="10144" length="5"/>
<text>ACBD3</text>
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<annotation id="252">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10201</offset>
<text>We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="10236" length="11"/>
<text>ACBD3:PI4KB</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10296" length="5"/>
<text>PI4KB</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:15:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10346" length="36"/>
<text>in vitro membrane recruitment system</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10389" length="26"/>
<text>Giant Unilamellar Vesicles</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10417" length="4"/>
<text>GUVs</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10438" length="5"/>
<text>PI4KB</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10460" length="2"/>
<text>PI</text>
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<annotation id="260">
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<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10487" length="5"/>
<text>PI4KB</text>
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<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10493" length="6"/>
<text>kinase</text>
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<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:06:50Z</infon>
<location offset="10517" length="9"/>
<text>localized</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10545" length="4"/>
<text>GUVs</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10609" length="5"/>
<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10637" length="4"/>
<text>GUVs</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10665" length="5"/>
<text>ACBD3</text>
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<annotation id="700">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:35:53Z</infon>
<location offset="10679" length="18"/>
<text>DGS-NTA (Ni) lipid</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10716" length="5"/>
<text>PI4KB</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10759</offset>
<text>We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10783" length="5"/>
<text>ACBD3</text>
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<annotation id="701">
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:36:14Z</infon>
<location offset="10814" length="20"/>
<text>localization signals</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10839" length="5"/>
<text>PI4KB</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:35:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10846" length="14"/>
<text>overexpression</text>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="10868" length="8"/>
<text>Q domain</text>
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<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10922" length="6"/>
<text>kinase</text>
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<infon key="score">0.9956189</infon>
<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10983" length="5"/>
<text>PI4KB</text>
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<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:36:55Z</infon>
<location offset="11018" length="14"/>
<text>overexpressing</text>
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<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:34:35Z</infon>
<location offset="11037" length="3"/>
<text>GFP</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="11043" length="8"/>
<text>Q domain</text>
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<annotation id="706">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:30Z</infon>
<location offset="11110" length="6"/>
<text>signal</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="11152" length="6"/>
<text>kinase</text>
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<infon key="identifier">DUMMY:</infon>
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<location offset="11264" length="6"/>
<text>signal</text>
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<infon key="updated_at">2023-09-21T15:35:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11279" length="14"/>
<text>overexpression</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:42:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11297" length="3"/>
<text>GFP</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:34:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11312" length="11"/>
<text>non-binding</text>
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<infon key="score">0.9050826</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11324" length="8"/>
<text>Q domain</text>
</annotation>
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<infon key="score">0.737417</infon>
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<infon key="updated_at">2023-09-21T15:38:02Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>mutant</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:36:44Z</infon>
<location offset="11361" length="12"/>
<text>localization</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11392" length="5"/>
<text>PI4KB</text>
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<annotation id="278">
<infon key="score">0.9735685</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:35:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11428" length="14"/>
<text>overexpression</text>
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<annotation id="655">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="11450" length="8"/>
<text>Q domain</text>
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<annotation id="279">
<infon key="score">0.99336</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11490" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="280">
<infon key="score">0.9975262</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11523" length="8"/>
<text>Ceramide</text>
</annotation>
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<infon key="score">0.52904665</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11584" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="282">
<infon key="score">0.81602323</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11622" length="21"/>
<text>fluorescently labeled</text>
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<infon key="score">0.998357</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11644" length="8"/>
<text>ceramide</text>
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<annotation id="705">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T18:50:57Z</infon>
<location offset="11716" length="14"/>
<text>overexpressing</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="11735" length="8"/>
<text>Q domain</text>
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<annotation id="284">
<infon key="score">0.998293</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11784" length="8"/>
<text>ceramide</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:37:01Z</infon>
<location offset="11819" length="10"/>
<text>expressing</text>
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<infon key="updated_at">2023-09-21T15:35:24Z</infon>
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<text>mutant</text>
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<text>Q domain</text>
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<text>ceramide</text>
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<text>We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).</text>
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<text>PI4KB</text>
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<text>fluorescently tagged</text>
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<infon key="updated_at">2023-09-21T15:38:29Z</infon>
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<text>FKBP12</text>
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<text>FK506 binding protein 12</text>
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<infon key="updated_at">2023-09-21T15:39:24Z</infon>
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<text>FRB</text>
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<infon key="updated_at">2023-09-21T17:21:55Z</infon>
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<text>fragment</text>
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<infon key="updated_at">2023-09-21T17:16:35Z</infon>
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<location offset="12286" length="4"/>
<text>mTOR</text>
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<infon key="updated_at">2023-09-21T17:18:35Z</infon>
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<text>rapamycin</text>
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<text>fused</text>
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<infon key="updated_at">2023-09-21T15:39:25Z</infon>
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<text>FRB</text>
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<infon key="updated_at">2023-09-21T15:40:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12350" length="5"/>
<text>34–63</text>
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<infon key="updated_at">2023-09-21T15:41:06Z</infon>
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<location offset="12363" length="33"/>
<text>mitochondrial localization signal</text>
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<infon key="updated_at">2023-09-21T15:39:34Z</infon>
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<location offset="12402" length="39"/>
<text>mitochondrial A-kinase anchor protein 1</text>
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<text>AKAP1</text>
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<infon key="updated_at">2023-09-21T15:41:30Z</infon>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="12469" length="8"/>
<text>Q domain</text>
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<infon key="updated_at">2023-09-21T16:15:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12487" length="8"/>
<text>fused to</text>
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<infon key="updated_at">2023-09-21T15:38:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12496" length="6"/>
<text>FKBP12</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:41:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12507" length="4"/>
<text>mRFP</text>
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<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:42:37Z</infon>
<location offset="12535" length="12"/>
<text>localization</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12555" length="5"/>
<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="12561" length="8"/>
<text>Q domain</text>
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<infon key="identifier">MESH:</infon>
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<infon key="updated_at">2023-09-21T15:39:53Z</infon>
<location offset="12574" length="3"/>
<text>GFP</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12580" length="5"/>
<text>PI4KB</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T17:18:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12619" length="9"/>
<text>rapamycin</text>
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<infon key="type">mutant</infon>
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<infon key="updated_at">2023-09-21T14:29:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12651" length="5"/>
<text>H284A</text>
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<annotation id="313">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:38:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12657" length="6"/>
<text>mutant</text>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12671" length="5"/>
<text>ACBD3</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="12677" length="8"/>
<text>Q domain</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12719" length="5"/>
<text>PI4KB</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="12725" length="6"/>
<text>kinase</text>
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<infon key="updated_at">2023-09-21T17:16:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12751" length="5"/>
<text>ACDB3</text>
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<location offset="12757" length="8"/>
<text>Q domain</text>
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<infon key="updated_at">2023-09-21T17:18:41Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>rapamycin</text>
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<infon key="updated_at">2023-09-21T15:40:10Z</infon>
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<text>wild-type</text>
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<location offset="12921" length="6"/>
<text>kinase</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<location offset="13013" length="5"/>
<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T16:15:37Z</infon>
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<text>co-expressed</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:40:09Z</infon>
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<infon key="updated_at">2023-09-21T17:16:55Z</infon>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="13067" length="8"/>
<text>Q domain</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="13103" length="12"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13148" length="5"/>
<text>PI4KB</text>
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<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:42:25Z</infon>
<location offset="13171" length="12"/>
<text>localization</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:15:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13189" length="12"/>
<text>co-expressed</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:40:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13211" length="15"/>
<text>non-interacting</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<location offset="13227" length="8"/>
<text>Q domain</text>
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<infon key="score">0.99899536</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:38:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13236" length="6"/>
<text>mutant</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
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<text>ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate</text>
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<infon key="identifier">PR:</infon>
<location offset="13272" length="5"/>
<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T15:44:10Z</infon>
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<text>enzymatic activity</text>
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<annotation id="329">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13327" length="5"/>
<text>PI4KB</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13368</offset>
<text>To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism.</text>
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<infon key="score">0.76173043</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13384" length="5"/>
<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13404" length="5"/>
<text>PI4KB</text>
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<annotation id="630">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="13410" length="6"/>
<text>kinase</text>
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<annotation id="720">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:10Z</infon>
<location offset="13417" length="18"/>
<text>enzymatic activity</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:43:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13460" length="24"/>
<text>luminescent kinase assay</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13491" length="2"/>
<text>PI</text>
</annotation>
<annotation id="631">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="13561" length="6"/>
<text>kinase</text>
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<annotation id="334">
<infon key="score">0.997044</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13580" length="5"/>
<text>PI4KB</text>
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<annotation id="335">
<infon key="score">0.78130734</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13612" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="336">
<infon key="score">0.8759795</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13714" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="337">
<infon key="score">0.9745813</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13783" length="5"/>
<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13821" length="2"/>
<text>PI</text>
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<annotation id="339">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:15:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13948" length="3"/>
<text>GUV</text>
</annotation>
<annotation id="340">
<infon key="score">0.9652853</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13964" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="740">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T16:06:50Z</infon>
<location offset="13970" length="9"/>
<text>localized</text>
</annotation>
<annotation id="341">
<infon key="score">0.9513302</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:43:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13987" length="3"/>
<text>GUV</text>
</annotation>
<annotation id="342">
<infon key="score">0.9741795</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14005" length="4"/>
<text>GUVs</text>
</annotation>
<annotation id="343">
<infon key="score">0.9987618</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14024" length="2"/>
<text>PI</text>
</annotation>
<annotation id="344">
<infon key="score">0.99452776</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14055" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="632">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="14061" length="6"/>
<text>kinase</text>
</annotation>
<annotation id="714">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:42:06Z</infon>
<location offset="14095" length="3"/>
<text>CFP</text>
</annotation>
<annotation id="718">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:43:18Z</infon>
<location offset="14099" length="4"/>
<text>SidC</text>
</annotation>
<annotation id="345">
<infon key="score">0.99893934</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14120" length="4"/>
<text>PI4P</text>
</annotation>
<annotation id="633">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="14184" length="6"/>
<text>kinase</text>
</annotation>
<annotation id="719">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:43:41Z</infon>
<location offset="14208" length="19"/>
<text>confocal microscope</text>
</annotation>
<annotation id="346">
<infon key="score">0.98986673</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14263" length="15"/>
<text>phosphorylation</text>
</annotation>
<annotation id="634">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="14295" length="6"/>
<text>kinase</text>
</annotation>
<annotation id="347">
<infon key="score">0.94844615</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T17:20:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14302" length="5"/>
<text>alone</text>
</annotation>
<annotation id="635">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<location offset="14321" length="6"/>
<text>kinase</text>
</annotation>
<annotation id="348">
<infon key="score">0.91498756</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14360" length="4"/>
<text>GUVs</text>
</annotation>
<annotation id="349">
<infon key="score">0.97361845</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14368" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="350">
<infon key="score">0.9978117</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14410" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="351">
<infon key="score">0.9976972</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:28Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14421" length="3"/>
<text>ATP</text>
</annotation>
<annotation id="352">
<infon key="score">0.9935861</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14488" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="724">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:11Z</infon>
<location offset="14494" length="18"/>
<text>enzymatic activity</text>
</annotation>
<annotation id="353">
<infon key="score">0.97472304</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14528" length="5"/>
<text>ACBD3</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>14664</offset>
<text>Discussion</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>14675</offset>
<text>Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.</text>
<annotation id="354">
<infon key="score">0.9349319</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14699" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="355">
<infon key="score">0.9901046</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14833" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="356">
<infon key="score">0.9934269</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14936" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="357">
<infon key="score">0.90053684</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15002" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="358">
<infon key="score">0.99780643</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15063" length="13"/>
<text>small GTPases</text>
</annotation>
<annotation id="359">
<infon key="score">0.9977786</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15077" length="5"/>
<text>Rab11</text>
</annotation>
<annotation id="360">
<infon key="score">0.99702436</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:06Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15087" length="4"/>
<text>Arf1</text>
</annotation>
<annotation id="361">
<infon key="score">0.9960869</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:02Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15112" length="36"/>
<text>acyl-CoA binding domain containing 3</text>
</annotation>
<annotation id="362">
<infon key="score">0.99874747</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15150" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="363">
<infon key="score">0.9988655</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15166" length="25"/>
<text>neuronal calcium sensor-1</text>
</annotation>
<annotation id="364">
<infon key="score">0.9990631</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:53Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15193" length="5"/>
<text>NCS-1</text>
</annotation>
<annotation id="365">
<infon key="score">0.9992748</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:19Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15213" length="9"/>
<text>frequenin</text>
</annotation>
<annotation id="366">
<infon key="score">0.9982541</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15226" length="5"/>
<text>yeast</text>
</annotation>
<annotation id="367">
<infon key="score">0.99290615</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:44:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15241" length="15"/>
<text>14-3-3 proteins</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>15258</offset>
<text>The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.</text>
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<infon key="score">0.9973226</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:01:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15262" length="9"/>
<text>monomeric</text>
</annotation>
<annotation id="369">
<infon key="score">0.99831736</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:01:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15272" length="9"/>
<text>G protein</text>
</annotation>
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<infon key="score">0.9986952</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15282" length="5"/>
<text>Rab11</text>
</annotation>
<annotation id="371">
<infon key="score">0.9975291</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:45:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15294" length="9"/>
<text>mammalian</text>
</annotation>
<annotation id="372">
<infon key="score">0.99633443</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15304" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="373">
<infon key="score">0.9992804</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
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<infon key="updated_at">2023-09-21T15:45:16Z</infon>
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<location offset="15487" length="4"/>
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<location offset="15503" length="25"/>
<text>small GTP binding protein</text>
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<text>PI4KB</text>
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<location offset="15706" length="9"/>
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<text>yeast</text>
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<text>PI4KB</text>
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<location offset="15874" length="5"/>
<text>PI4KB</text>
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<text>PI4KB</text>
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<text>phosphorylation</text>
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<location offset="16285" length="5"/>
<text>PI4KB</text>
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<text>active</text>
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<infon key="identifier">MESH:</infon>
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<text>PI4KB</text>
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</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>16782</offset>
<text>Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.</text>
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<infon key="updated_at">2023-09-21T14:07:52Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>Kd</text>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<infon key="updated_at">2023-09-21T13:55:14Z</infon>
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<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T16:15:59Z</infon>
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<infon key="updated_at">2023-09-21T15:46:55Z</infon>
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<infon key="updated_at">2023-09-21T13:56:16Z</infon>
<infon key="identifier">GO:</infon>
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<text>ACBD3:PI4KB</text>
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<infon key="score">0.9979705</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17253" length="5"/>
<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T15:47:13Z</infon>
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<infon key="updated_at">2023-09-21T17:22:21Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>44–64</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-21T15:47:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17381" length="20"/>
<text>helical hairpin fold</text>
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<infon key="updated_at">2023-09-21T14:25:42Z</infon>
<infon key="identifier">SO:</infon>
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<text>kinase helix</text>
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<infon key="score">0.8986097</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<infon key="identifier">MESH:</infon>
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<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
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<text>ACBD3</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<infon key="identifier">MESH:</infon>
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<text>kinase</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:04:05Z</infon>
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<text>kinase</text>
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<infon key="updated_at">2023-09-21T14:04:05Z</infon>
<infon key="identifier">MESH:</infon>
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<text>kinase</text>
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<infon key="updated_at">2023-09-21T14:16:05Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>structures</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:47:26Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>pseudoatomic model</text>
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<infon key="score">0.99829084</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17937" length="5"/>
<text>PI4KB</text>
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<infon key="identifier">PR:</infon>
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<text>Rab11</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
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<text>+RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.</text>
<annotation id="433">
<infon key="score">0.9262333</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:47:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18118" length="12"/>
<text>+RNA viruses</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:52Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>PI4P</text>
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<infon key="score">0.9953243</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:57:20Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>viral</text>
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<annotation id="436">
<infon key="score">0.99788505</infon>
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<infon key="updated_at">2023-09-21T15:48:00Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>viruses</text>
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<annotation id="614">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:38Z</infon>
<location offset="18437" length="4"/>
<text>PI4K</text>
</annotation>
<annotation id="620">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:59:38Z</infon>
<location offset="18442" length="7"/>
<text>kinases</text>
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<infon key="updated_at">2023-09-21T13:55:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18480" length="4"/>
<text>PI4P</text>
</annotation>
<annotation id="438">
<infon key="score">0.699083</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T17:18:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18485" length="5"/>
<text>lipid</text>
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<annotation id="759">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T17:21:20Z</infon>
<location offset="18492" length="26"/>
<text>Non-structural 3A proteins</text>
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<annotation id="439">
<infon key="score">0.99718</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:03:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18529" length="14"/>
<text>picornaviruses</text>
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<annotation id="440">
<infon key="score">0.9856564</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:48:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18553" length="11"/>
<text>Enterovirus</text>
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<annotation id="441">
<infon key="score">0.99433774</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:48:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18571" length="10"/>
<text>poliovirus</text>
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<annotation id="725">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:49:06Z</infon>
<location offset="18583" length="17"/>
<text>coxsackievirus-B3</text>
</annotation>
<annotation id="726">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:49:20Z</infon>
<location offset="18602" length="13"/>
<text>rhinovirus-14</text>
</annotation>
<annotation id="442">
<infon key="score">0.7567454</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:49:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18621" length="9"/>
<text>Kobuvirus</text>
</annotation>
<annotation id="443">
<infon key="score">0.9271165</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:49:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18637" length="13"/>
<text>Aichi virus-1</text>
</annotation>
<annotation id="444">
<infon key="score">0.99703985</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18682" length="5"/>
<text>ACBD3</text>
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<annotation id="445">
<infon key="score">0.99915993</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:49:48Z</infon>
<infon key="identifier">GO:</infon>
<location offset="18734" length="14"/>
<text>3A:ACBD3:PI4KB</text>
</annotation>
<annotation id="446">
<infon key="score">0.99662316</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18772" length="9"/>
<text>structure</text>
</annotation>
<annotation id="447">
<infon key="score">0.99881554</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18789" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="448">
<infon key="score">0.9772059</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:06:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18795" length="8"/>
<text>Q domain</text>
</annotation>
<annotation id="449">
<infon key="score">0.99930453</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:25:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18812" length="12"/>
<text>kinase helix</text>
</annotation>
<annotation id="450">
<infon key="score">0.9953868</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18905" length="5"/>
<text>ACBD3</text>
</annotation>
<annotation id="451">
<infon key="score">0.9890894</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18912" length="5"/>
<text>PI4KB</text>
</annotation>
<annotation id="452">
<infon key="score">0.99866444</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:56:17Z</infon>
<infon key="identifier">GO:</infon>
<location offset="18927" length="11"/>
<text>ACBD3:PI4KB</text>
</annotation>
<annotation id="453">
<infon key="score">0.9974644</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18954" length="12"/>
<text>picornaviral</text>
</annotation>
<annotation id="454">
<infon key="score">0.9958832</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:03:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19059" length="14"/>
<text>picornaviruses</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>19131</offset>
<text>Materials and Methods</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>19153</offset>
<text>Plasmid construction, protein expression, and purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>19212</offset>
<text>All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>20638</offset>
<text>In vitro pull-downs</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>20658</offset>
<text>Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>21143</offset>
<text>SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation)</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>21216</offset>
<text>PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations:</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>21600</offset>
<text>where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>21856</offset>
<text>AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>22360</offset>
<text>In vitro kinase assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>22382</offset>
<text>In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>23046</offset>
<text>NMR spectroscopy</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>23063</offset>
<text>NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>23877</offset>
<text>The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>24170</offset>
<text>Protein labeling with fluorescent dyes</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>24209</offset>
<text>PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>24530</offset>
<text>Giant Unilamellar Vesicle Preparation and Imaging</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>24580</offset>
<text>Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>24979</offset>
<text>Live Cell Imaging</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>24997</offset>
<text>COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>25988</offset>
<text>Immunofluorescent imaging</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>26014</offset>
<text>COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>26738</offset>
<text>HD exchange</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>26750</offset>
<text>Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>27188</offset>
<text>Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>28355</offset>
<text>Additional Information</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28378</offset>
<text>Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28614</offset>
<text>How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. Rep. 6, 23641; doi: 10.1038/srep23641 (2016).</text>
</passage>
<passage>
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<text>Author Contributions M.K. and A.D. carried out DNA cloning, M.K., A.B., D.C. and E.B. carried out protein expression and purification, M.K. performed pull-down assays, L.R. carried out analytical ultracentrifugation, M.K. and J.T. performed S.P.R. experiments, R.H. and V.V. carried out NMR experiments, structure refinement, and deposition, A.B. and P.M. performed HDX/MS experiments, D.C. carried out in vitro kinase assay, E.B. performed protein labeling, E.B. and J.H. carried out GUV preparation and imaging, D.T. and N.S. performed some of the cloning and the cell-based experiments, E.B. supervised the project, E.B., M.K., M.N., V.V. and T.B. wrote the manuscript, all authors contributed to data analysis and commented on the manuscript.</text>
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<passage>
<infon key="file">srep23641-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>34270</offset>
<text>Biochemical characterization of the ACBD3:PI4KB complex.</text>
<annotation id="727">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T15:58:09Z</infon>
<location offset="34270" length="28"/>
<text>Biochemical characterization</text>
</annotation>
<annotation id="455">
<infon key="score">0.99930733</infon>
<infon key="type">complex_assembly</infon>
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<infon key="updated_at">2023-09-21T13:56:17Z</infon>
<infon key="identifier">GO:</infon>
<location offset="34306" length="11"/>
<text>ACBD3:PI4KB</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23641-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>34327</offset>
<text>(A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.</text>
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<infon key="identifier">PR:</infon>
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<text>ACBD3</text>
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<text>PI4KB</text>
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<infon key="identifier">PR:</infon>
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<text>ACBD3</text>
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<text>acyl-CoA binding domain</text>
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<text>ACBD</text>
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<infon key="updated_at">2023-09-21T15:58:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34467" length="26"/>
<text>charged amino acids region</text>
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<infon key="updated_at">2023-09-21T15:58:33Z</infon>
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<location offset="34495" length="3"/>
<text>CAR</text>
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<infon key="updated_at">2023-09-21T15:58:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34501" length="21"/>
<text>glutamine rich region</text>
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<infon key="score">0.9982855</infon>
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<infon key="updated_at">2023-09-21T15:58:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34524" length="1"/>
<text>Q</text>
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<infon key="score">0.9983718</infon>
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<infon key="updated_at">2023-09-21T15:58:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34532" length="21"/>
<text>Golgi dynamics domain</text>
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<infon key="updated_at">2023-09-21T15:58:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34555" length="4"/>
<text>GOLD</text>
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<infon key="score">0.9927367</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="34562" length="5"/>
<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T14:08:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34587" length="17"/>
<text>N-terminal region</text>
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<annotation id="469">
<infon key="score">0.999196</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T14:05:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34606" length="14"/>
<text>helical domain</text>
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<infon key="score">0.9976603</infon>
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<infon key="updated_at">2023-09-21T15:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34626" length="13"/>
<text>kinase domain</text>
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<infon key="score">0.9992599</infon>
<infon key="type">structure_element</infon>
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<infon key="updated_at">2023-09-21T15:59:01Z</infon>
<infon key="identifier">SO:</infon>
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<text>N- and C-terminal lobes</text>
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<infon key="score">0.9987996</infon>
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<infon key="updated_at">2023-09-21T15:59:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34695" length="24"/>
<text>In vitro pull-down assay</text>
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<infon key="score">0.9987951</infon>
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<infon key="updated_at">2023-09-21T15:59:07Z</infon>
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<text>Pull-down assays</text>
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<infon key="identifier">DUMMY:</infon>
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<location offset="34788" length="14"/>
<text>His6GB1-tagged</text>
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<infon key="identifier">DUMMY:</infon>
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<text>untagged</text>
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<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-21T15:59:29Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>full-length</text>
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<infon key="score">0.99147826</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:55:14Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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<text>ACBD3</text>
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<text>SDS gels</text>
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<infon key="updated_at">2023-09-21T14:06:47Z</infon>
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<text>Analytical Ultracentrifugation</text>
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<infon key="score">0.99885726</infon>
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<infon key="identifier">MESH:</infon>
<location offset="35170" length="3"/>
<text>AUC</text>
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<infon key="identifier">GO:</infon>
<location offset="35190" length="11"/>
<text>ACBD3:PI4KB</text>
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<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-21T16:00:25Z</infon>
<location offset="35283" length="39"/>
<text>ACBD3 Q domain: PI4KB N terminal region</text>
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<infon key="score">0.9989316</infon>
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<infon key="updated_at">2023-09-21T14:07:05Z</infon>
<infon key="identifier">MESH:</infon>
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<text>Surface plasmon resonance</text>
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<text>SPR</text>
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<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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<passage>
<infon key="file">srep23641-f2.jpg</infon>
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<infon key="section_type">FIG</infon>
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<text>(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. ACBD3 is shown in magenta and PI4KB in orange. (C) Top view of the kinase helix. The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. Inputs and bound proteins were analyzed on SDS gels and stained with Coomassie Blue. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels.</text>
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<infon key="updated_at">2023-09-21T14:16:05Z</infon>
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<text>structures</text>
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<location offset="35753" length="8"/>
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<infon key="updated_at">2023-09-21T14:16:05Z</infon>
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<text>structures</text>
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<annotation id="497">
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<infon key="updated_at">2023-09-21T16:01:36Z</infon>
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<text>folded</text>
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<infon key="updated_at">2023-09-21T13:55:15Z</infon>
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<location offset="35864" length="5"/>
<text>PI4KB</text>
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<annotation id="499">
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<infon key="updated_at">2023-09-21T14:27:41Z</infon>
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<text>hydrogen bonds</text>
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<infon key="updated_at">2023-09-21T13:55:21Z</infon>
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<location offset="36008" length="5"/>
<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T14:27:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="36014" length="6"/>
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<infon key="updated_at">2023-09-21T13:55:15Z</infon>
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<location offset="36022" length="5"/>
<text>PI4KB</text>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T13:55:15Z</infon>
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<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T14:26:44Z</infon>
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<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T14:06:07Z</infon>
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<location offset="36145" length="5"/>
<text>ACBD3</text>
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<infon key="updated_at">2023-09-21T13:55:15Z</infon>
<infon key="identifier">PR:</infon>
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<text>PI4KB</text>
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<infon key="updated_at">2023-09-21T14:25:42Z</infon>
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<text>kinase helix</text>
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<infon key="updated_at">2023-09-21T14:25:42Z</infon>
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<text>amphipathic</text>
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<text>ACBD3</text>
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<text>Pull-down assay</text>
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<infon key="updated_at">2023-09-21T14:05:17Z</infon>
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<text>ACBD3</text>
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<text>ACBD3 is sufficient to recruit the PI4KB kinase to membranes.</text>
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<text>ACBD3</text>
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<text>kinase</text>
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<infon key="file">srep23641-f3.jpg</infon>
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<infon key="section_type">FIG</infon>
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<text>(A) GUVs recruitment assay. Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. (B) Golgi displacement experiment. Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. Middle panel: The same experiment performed with GFP alone. Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. Middle panel – The same experiment performed with mRFP-FKBP alone. Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. Right – The same experiment performed using the H264A Q domain mutant.</text>
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<text>(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. The error bars stand for SEM based on three independent experiments (also SI Fig. 6).</text>
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<text>PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.</text>
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<id>4817029</id>
<infon key="license">CC BY</infon>
<infon key="tt_curatable">no</infon>
<infon key="tt_version">2</infon>
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<passage>
<infon key="article-id_doi">10.1038/srep23473</infon>
<infon key="article-id_pii">srep23473</infon>
<infon key="article-id_pmc">4817029</infon>
<infon key="article-id_pmid">27032335</infon>
<infon key="elocation-id">23473</infon>
<infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
<infon key="name_0">surname:Liberato;given-names:Marcelo V.</infon>
<infon key="name_1">surname:Silveira;given-names:Rodrigo L.</infon>
<infon key="name_10">surname:Polikarpov;given-names:Igor</infon>
<infon key="name_2">surname:Prates;given-names:Érica T.</infon>
<infon key="name_3">surname:de Araujo;given-names:Evandro A.</infon>
<infon key="name_4">surname:Pellegrini;given-names:Vanessa O. A.</infon>
<infon key="name_5">surname:Camilo;given-names:Cesar M.</infon>
<infon key="name_6">surname:Kadowaki;given-names:Marco A.</infon>
<infon key="name_7">surname:Neto;given-names:Mario de O.</infon>
<infon key="name_8">surname:Popov;given-names:Alexander</infon>
<infon key="name_9">surname:Skaf;given-names:Munir S.</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">6</infon>
<infon key="year">2016</infon>
<offset>0</offset>
<text>Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism</text>
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<text>family 5 glycoside hydrolase</text>
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<infon key="section_type">ABSTRACT</infon>
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<text>Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.</text>
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<text>Glycoside hydrolases</text>
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<text>GHs</text>
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<infon key="updated_at">2023-09-21T09:40:08Z</infon>
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<text>full-length</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="241" length="9"/>
<text>structure</text>
</annotation>
<annotation id="5">
<infon key="score">0.9992067</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="256" length="9"/>
<text>cellulase</text>
</annotation>
<annotation id="6">
<infon key="score">0.99826276</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="271" length="22"/>
<text>Bacillus licheniformis</text>
</annotation>
<annotation id="7">
<infon key="score">0.99894685</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="295" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="8">
<infon key="score">0.99763244</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="321" length="15"/>
<text>GH5 subfamily 4</text>
</annotation>
<annotation id="9">
<infon key="score">0.9979503</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="375" length="17"/>
<text>ancillary modules</text>
</annotation>
<annotation id="10">
<infon key="score">0.99924576</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="394" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="11">
<infon key="score">0.9989945</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="413" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="12">
<infon key="score">0.99890447</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="450" length="21"/>
<text>X-ray crystallography</text>
</annotation>
<annotation id="13">
<infon key="score">0.99891025</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="473" length="28"/>
<text>small-angle X-ray scattering</text>
</annotation>
<annotation id="14">
<infon key="score">0.9987839</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="506" length="30"/>
<text>molecular dynamics simulations</text>
</annotation>
<annotation id="15">
<infon key="score">0.9945221</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="569" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="16">
<infon key="score">0.99930584</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="602" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="17">
<infon key="score">0.99936837</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="620" length="2"/>
<text>CD</text>
</annotation>
<annotation id="18">
<infon key="score">0.9930973</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="639" length="16"/>
<text>fully functional</text>
</annotation>
<annotation id="19">
<infon key="score">0.999096</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="656" length="11"/>
<text>active site</text>
</annotation>
<annotation id="20">
<infon key="score">0.9993052</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="770" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="21">
<infon key="score">0.98631513</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="834" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="22">
<infon key="score">0.99929404</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="852" length="2"/>
<text>CD</text>
</annotation>
<annotation id="23">
<infon key="score">0.9990971</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="878" length="15"/>
<text>binding subsite</text>
</annotation>
<annotation id="24">
<infon key="score">0.9990859</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="938" length="2"/>
<text>GH</text>
</annotation>
<annotation id="25">
<infon key="score">0.99607617</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="983" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="26">
<infon key="score">0.9482441</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1009" length="23"/>
<text>catalytically competent</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1053</offset>
<text>The production of biofuels from renewable sources is an important element of the global strategy for generating sustainable energy with reduced environmental impact. Current technologies for obtaining liquid biofuels and green chemicals rely on the enzymatic digestion of lignocellulosic biomass from a variety of feedstocks. Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin. Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.</text>
<annotation id="27">
<infon key="score">0.9980286</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1379" length="5"/>
<text>Plant</text>
</annotation>
<annotation id="28">
<infon key="score">0.9926569</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1421" length="13"/>
<text>carbohydrates</text>
</annotation>
<annotation id="798">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:00Z</infon>
<location offset="1469" length="9"/>
<text>cellulose</text>
</annotation>
<annotation id="29">
<infon key="score">0.75858444</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1543" length="13"/>
<text>hemicellulose</text>
</annotation>
<annotation id="30">
<infon key="score">0.8075788</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1561" length="6"/>
<text>lignin</text>
</annotation>
<annotation id="31">
<infon key="score">0.9981589</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1569" length="5"/>
<text>Plant</text>
</annotation>
<annotation id="32">
<infon key="score">0.98998487</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1688" length="6"/>
<text>sugars</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1696</offset>
<text>The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes. Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose. Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically. The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.</text>
<annotation id="33">
<infon key="score">0.6507011</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1745" length="9"/>
<text>cellulose</text>
</annotation>
<annotation id="34">
<infon key="score">0.9988967</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1799" length="18"/>
<text>Cellobiohydrolases</text>
</annotation>
<annotation id="35">
<infon key="score">0.9992205</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1819" length="4"/>
<text>CBHs</text>
</annotation>
<annotation id="36">
<infon key="score">0.877582</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1907" length="9"/>
<text>cellulose</text>
</annotation>
<annotation id="37">
<infon key="score">0.99929</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:53:00Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1958" length="10"/>
<text>cellobiose</text>
</annotation>
<annotation id="38">
<infon key="score">0.99881196</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:45:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1970" length="14"/>
<text>Endoglucanases</text>
</annotation>
<annotation id="39">
<infon key="score">0.9992318</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1986" length="3"/>
<text>EGs</text>
</annotation>
<annotation id="40">
<infon key="score">0.7187887</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2041" length="9"/>
<text>cellulose</text>
</annotation>
<annotation id="41">
<infon key="score">0.81927955</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2088" length="3"/>
<text>CBH</text>
</annotation>
<annotation id="42">
<infon key="score">0.9992581</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:53:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2154" length="10"/>
<text>cellobiose</text>
</annotation>
<annotation id="43">
<infon key="score">0.9990037</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2213" length="7"/>
<text>glucose</text>
</annotation>
<annotation id="44">
<infon key="score">0.99811155</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2224" length="14"/>
<text>β-glucosidases</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2243</offset>
<text>The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers. The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains. Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands. CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood. Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.</text>
<annotation id="45">
<infon key="score">0.99880946</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:39:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2273" length="20"/>
<text>glycoside hydrolases</text>
</annotation>
<annotation id="46">
<infon key="score">0.9992569</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2295" length="3"/>
<text>GHs</text>
</annotation>
<annotation id="47">
<infon key="score">0.9993674</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2325" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="48">
<infon key="score">0.99939394</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2343" length="2"/>
<text>CD</text>
</annotation>
<annotation id="49">
<infon key="score">0.99907243</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2389" length="17"/>
<text>ancillary modules</text>
</annotation>
<annotation id="50">
<infon key="score">0.99794024</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2408" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="51">
<infon key="score">0.81550145</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2445" length="15"/>
<text>less structured</text>
</annotation>
<annotation id="52">
<infon key="score">0.99828774</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2461" length="7"/>
<text>linkers</text>
</annotation>
<annotation id="53">
<infon key="score">0.97873074</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2494" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="54">
<infon key="score">0.99920225</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2502" length="28"/>
<text>carbohydrate-binding modules</text>
</annotation>
<annotation id="55">
<infon key="score">0.99862635</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2532" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="56">
<infon key="score">0.99761057</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2585" length="12"/>
<text>carbohydrate</text>
</annotation>
<annotation id="57">
<infon key="score">0.9987066</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2665" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="58">
<infon key="score">0.98361444</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2681" length="12"/>
<text>carbohydrate</text>
</annotation>
<annotation id="59">
<infon key="score">0.9992988</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2816" length="2"/>
<text>CD</text>
</annotation>
<annotation id="60">
<infon key="score">0.9990503</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2853" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="61">
<infon key="score">0.76988083</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2906" length="9"/>
<text>cellulose</text>
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<annotation id="62">
<infon key="score">0.9990746</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2997" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="63">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3031" length="3"/>
<text>CDs</text>
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<annotation id="64">
<infon key="score">0.99435884</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3038" length="12"/>
<text>carbohydrate</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3205</offset>
<text>Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity. In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B). A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase. Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.</text>
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<infon key="score">0.99921894</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:57Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>active</text>
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<annotation id="66">
<infon key="score">0.99879116</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3243" length="3"/>
<text>GHs</text>
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<annotation id="67">
<infon key="score">0.99908733</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3252" length="4"/>
<text>lack</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3257" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="69">
<infon key="score">0.99507636</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3300" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="70">
<infon key="score">0.9975841</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3336" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="71">
<infon key="score">0.99947864</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3381" length="2"/>
<text>CD</text>
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<annotation id="72">
<infon key="score">0.9988356</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3384" length="22"/>
<text>substrate-binding site</text>
</annotation>
<annotation id="73">
<infon key="score">0.99826276</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3423" length="27"/>
<text>carbohydrate-active enzymes</text>
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<annotation id="74">
<infon key="score">0.9975892</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3460" length="17"/>
<text>endo/exocellulase</text>
</annotation>
<annotation id="75">
<infon key="score">0.2739183</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:52Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3478" length="2"/>
<text>E4</text>
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<annotation id="76">
<infon key="score">0.99860615</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3486" length="18"/>
<text>Thermobifida fusca</text>
</annotation>
<annotation id="77">
<infon key="score">0.9587691</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:02Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3506" length="11"/>
<text>chitinase B</text>
</annotation>
<annotation id="78">
<infon key="score">0.99851334</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3523" length="19"/>
<text>Serratia marcescens</text>
</annotation>
<annotation id="79">
<infon key="score">0.99893034</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3546" length="18"/>
<text>starch phosphatase</text>
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<annotation id="80">
<infon key="score">0.9970911</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3570" length="20"/>
<text>Arabidopsis thaliana</text>
</annotation>
<annotation id="81">
<infon key="score">0.8692663</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3597" length="15"/>
<text>GH5 subfamily 4</text>
</annotation>
<annotation id="808">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="3614" length="5"/>
<text>GH5_4</text>
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<annotation id="82">
<infon key="score">0.99865353</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3621" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="83">
<infon key="score">0.99876606</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3640" length="19"/>
<text>Bacillus halodurans</text>
</annotation>
<annotation id="84">
<infon key="score">0.9987307</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3661" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="85">
<infon key="score">0.9760523</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3750" length="3"/>
<text>GH9</text>
</annotation>
<annotation id="86">
<infon key="score">0.99953926</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3754" length="2"/>
<text>CD</text>
</annotation>
<annotation id="820">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:46Z</infon>
<location offset="3762" length="11"/>
<text>type C CBM3</text>
</annotation>
<annotation id="87">
<infon key="score">0.99915934</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3819" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="88">
<infon key="score">0.9645166</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3867" length="3"/>
<text>CBM</text>
</annotation>
<annotation id="89">
<infon key="score">0.99893147</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3906" length="11"/>
<text>active site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3974</offset>
<text>Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module. Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis. Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings. Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate. It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution. It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.</text>
<annotation id="90">
<infon key="score">0.99868464</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4013" length="15"/>
<text>X-ray structure</text>
</annotation>
<annotation id="91">
<infon key="score">0.9566901</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4036" length="11"/>
<text>tri-modular</text>
</annotation>
<annotation id="809">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="4048" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="92">
<infon key="score">0.9982936</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4054" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="93">
<infon key="score">0.9985973</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4073" length="19"/>
<text>Bacillus halodurans</text>
</annotation>
<annotation id="94">
<infon key="score">0.9991042</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4119" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="796">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<location offset="4138" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="95">
<infon key="score">0.9987675</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4161" length="11"/>
<text>active site</text>
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<annotation id="96">
<infon key="score">0.9993994</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4189" length="2"/>
<text>CD</text>
</annotation>
<annotation id="97">
<infon key="score">0.999167</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4199" length="31"/>
<text>immunoglobulin (Ig)-like module</text>
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<annotation id="98">
<infon key="score">0.9758551</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4232" length="10"/>
<text>Removal of</text>
</annotation>
<annotation id="99">
<infon key="score">0.9956642</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4247" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="100">
<infon key="score">0.99816173</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4319" length="9"/>
<text>β-glucans</text>
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<annotation id="101">
<infon key="score">0.9977476</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:58Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4369" length="10"/>
<text>xyloglucan</text>
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<annotation id="102">
<infon key="score">0.9944535</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4406" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="103">
<infon key="score">0.9991316</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4451" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="104">
<infon key="score">0.99774253</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4471" length="5"/>
<text>plant</text>
</annotation>
<annotation id="105">
<infon key="score">0.9915229</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:50:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4497" length="8"/>
<text>Modeling</text>
</annotation>
<annotation id="106">
<infon key="score">0.9992712</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:50:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4509" length="11"/>
<text>cellotriose</text>
</annotation>
<annotation id="107">
<infon key="score">0.9989642</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:50:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4528" length="17"/>
<text>negative subsites</text>
</annotation>
<annotation id="108">
<infon key="score">0.9991369</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4553" length="11"/>
<text>active site</text>
</annotation>
<annotation id="109">
<infon key="score">0.9991585</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4568" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="110">
<infon key="score">0.9542444</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:50:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4593" length="23"/>
<text>structural conservation</text>
</annotation>
<annotation id="111">
<infon key="score">0.8204445</infon>
<infon key="type">residue_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:50:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4624" length="2"/>
<text>-1</text>
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<annotation id="112">
<infon key="score">0.98499066</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4703" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="113">
<infon key="score">0.99886227</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:51:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4769" length="8"/>
<text>β-glucan</text>
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<annotation id="114">
<infon key="score">0.99476916</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4818" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="115">
<infon key="score">0.99862576</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4872" length="10"/>
<text>xyloglucan</text>
</annotation>
<annotation id="116">
<infon key="score">0.9993088</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4920" length="2"/>
<text>CD</text>
</annotation>
<annotation id="117">
<infon key="score">0.9971175</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4969" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="118">
<infon key="score">0.9991604</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5004" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="119">
<infon key="score">0.9982805</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:51:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5020" length="11"/>
<text>xyloglucans</text>
</annotation>
<annotation id="120">
<infon key="score">0.9978167</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5073" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="121">
<infon key="score">0.9991603</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5122" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="122">
<infon key="score">0.99331254</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:51:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5138" length="23"/>
<text>xyloglucan-rich regions</text>
</annotation>
<annotation id="123">
<infon key="score">0.99859613</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5184" length="5"/>
<text>plant</text>
</annotation>
<annotation id="124">
<infon key="score">0.9228424</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5255" length="3"/>
<text>AMs</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>5349</offset>
<text>The mechanisms of ligand binding mediated by large-scale conformational changes in proteins following the induced-fit or conformational selection models have recently attracted considerable attention. Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model. While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.</text>
<annotation id="858">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<location offset="5695" length="8"/>
<text>extended</text>
</annotation>
<annotation id="125">
<infon key="score">0.99613124</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:51:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5813" length="27"/>
<text>carbohydrate-active enzymes</text>
</annotation>
<annotation id="126">
<infon key="score">0.999311</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:45:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5995" length="14"/>
<text>endoglucanases</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>6011</offset>
<text>Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD. We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B. We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis. We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.</text>
<annotation id="127">
<infon key="score">0.9939641</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6031" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="128">
<infon key="score">0.99908876</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6054" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="810">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="6066" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="129">
<infon key="score">0.9983735</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6084" length="22"/>
<text>Bacillus licheniformis</text>
</annotation>
<annotation id="130">
<infon key="score">0.99898714</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6108" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="131">
<infon key="score">0.9986204</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6135" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="132">
<infon key="score">0.9992919</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6140" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="133">
<infon key="score">0.998767</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6159" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="134">
<infon key="score">0.9994461</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6182" length="2"/>
<text>CD</text>
</annotation>
<annotation id="135">
<infon key="score">0.9962609</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6189" length="42"/>
<text>structurally and functionally characterize</text>
</annotation>
<annotation id="136">
<infon key="score">0.99858046</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6266" length="23"/>
<text>protein crystallography</text>
</annotation>
<annotation id="137">
<infon key="score">0.9989625</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6291" length="28"/>
<text>small-angle X-ray scattering</text>
</annotation>
<annotation id="138">
<infon key="score">0.99831426</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6321" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="139">
<infon key="score">0.99881864</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6328" length="39"/>
<text>molecular dynamics computer simulations</text>
</annotation>
<annotation id="140">
<infon key="score">0.9988634</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6372" length="25"/>
<text>site-directed mutagenesis</text>
</annotation>
<annotation id="141">
<infon key="score">0.99568814</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6417" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="142">
<infon key="score">0.9990846</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6499" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="143">
<infon key="score">0.99801886</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6578" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="144">
<infon key="score">0.9992043</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6600" length="20"/>
<text>Ig-like hinge domain</text>
</annotation>
<annotation id="145">
<infon key="score">0.99893785</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6636" length="11"/>
<text>active-site</text>
</annotation>
<annotation id="146">
<infon key="score">0.99904066</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6723" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="811">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="6844" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="147">
<infon key="score">0.9993019</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6906" length="3"/>
<text>GHs</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>6911</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>6919</offset>
<text>BlCel5B Crystal Structure</text>
<annotation id="148">
<infon key="score">0.9960616</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6919" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="149">
<infon key="score">0.99812376</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6927" length="17"/>
<text>Crystal Structure</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>6945</offset>
<text>BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1). The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate). Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed. A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.</text>
<annotation id="150">
<infon key="score">0.99899524</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6945" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="151">
<infon key="score">0.99850273</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6953" length="8"/>
<text>crystals</text>
</annotation>
<annotation id="152">
<infon key="score">0.9985334</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6969" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="153">
<infon key="score">0.99859357</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6993" length="14"/>
<text>complexed with</text>
</annotation>
<annotation id="154">
<infon key="score">0.9993591</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7008" length="13"/>
<text>cellopentaose</text>
</annotation>
<annotation id="155">
<infon key="score">0.9694119</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7023" length="2"/>
<text>C5</text>
</annotation>
<annotation id="156">
<infon key="score">0.9989707</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7131" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="157">
<infon key="score">0.99924445</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7150" length="9"/>
<text>complexed</text>
</annotation>
<annotation id="158">
<infon key="score">0.9981719</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7160" length="10"/>
<text>structures</text>
</annotation>
<annotation id="159">
<infon key="score">0.9984168</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7297" length="5"/>
<text>loops</text>
</annotation>
<annotation id="160">
<infon key="score">0.9989159</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7325" length="22"/>
<text>substrate-binding site</text>
</annotation>
<annotation id="161">
<infon key="score">0.9982372</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7351" length="27"/>
<text>root mean squared deviation</text>
</annotation>
<annotation id="162">
<infon key="score">0.9980987</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7380" length="4"/>
<text>rmsd</text>
</annotation>
<annotation id="163">
<infon key="score">0.9992434</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7408" length="9"/>
<text>complexed</text>
</annotation>
<annotation id="164">
<infon key="score">0.9989364</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7422" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="165">
<infon key="score">0.9980007</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7437" length="10"/>
<text>structures</text>
</annotation>
<annotation id="166">
<infon key="score">0.94741315</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7578" length="17"/>
<text>first 17 residues</text>
</annotation>
<annotation id="167">
<infon key="score">0.99886227</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7613" length="4"/>
<text>loop</text>
</annotation>
<annotation id="168">
<infon key="score">0.99858093</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7626" length="4"/>
<text>L398</text>
</annotation>
<annotation id="169">
<infon key="score">0.9983217</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7635" length="4"/>
<text>P405</text>
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<annotation id="170">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7652" length="16"/>
<text>electron density</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>7679</offset>
<text>The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B). Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices. Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively. Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C). A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2. However, despite their structural resemblance, these modules share only 17% sequence identity. A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3. The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4. The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).</text>
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<infon key="identifier">PR:</infon>
<location offset="7683" length="7"/>
<text>BlCel5B</text>
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<text>structure</text>
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<text>CD</text>
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<infon key="updated_at">2023-09-21T09:54:45Z</infon>
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<text>18 to 330</text>
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<infon key="updated_at">2023-09-21T09:40:52Z</infon>
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<infon key="updated_at">2023-09-21T09:54:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7802" length="10"/>
<text>335 to 428</text>
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<infon key="updated_at">2023-09-21T09:55:07Z</infon>
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<location offset="7820" length="13"/>
<text>family 46 CBM</text>
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<annotation id="178">
<infon key="score">0.9974942</infon>
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<infon key="updated_at">2023-09-21T09:54:55Z</infon>
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<text>432 to 533</text>
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<annotation id="804">
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<infon key="identifier">MESH:</infon>
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<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<location offset="7903" length="3"/>
<text>GH5</text>
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<infon key="updated_at">2023-09-21T09:41:26Z</infon>
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<location offset="7919" length="2"/>
<text>CD</text>
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<location offset="7925" length="7"/>
<text>BlCel5B</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:55:23Z</infon>
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<location offset="7947" length="15"/>
<text>TIM barrel fold</text>
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<annotation id="182">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:55:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7980" length="9"/>
<text>β-strands</text>
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<annotation id="183">
<infon key="score">0.99879986</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:55:32Z</infon>
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<location offset="8006" length="9"/>
<text>α helices</text>
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<annotation id="184">
<infon key="score">0.9226088</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:47Z</infon>
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<location offset="8043" length="5"/>
<text>loops</text>
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<annotation id="821">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:55:33Z</infon>
<location offset="8065" length="9"/>
<text>α helices</text>
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<annotation id="799">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:59Z</infon>
<location offset="8087" length="7"/>
<text>linkers</text>
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<annotation id="185">
<infon key="score">0.9010072</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:56:00Z</infon>
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<location offset="8096" length="14"/>
<text>D429-D430-P431</text>
</annotation>
<annotation id="822">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:03Z</infon>
<location offset="8115" length="19"/>
<text>V331-P332-N333-A334</text>
</annotation>
<annotation id="186">
<infon key="score">0.99080867</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8148" length="5"/>
<text>CBM46</text>
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<annotation id="187">
<infon key="score">0.9991174</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8161" length="14"/>
<text>Ig-like module</text>
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<annotation id="188">
<infon key="score">0.99914384</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8184" length="14"/>
<text>Ig-like module</text>
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<annotation id="189">
<infon key="score">0.99945945</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8206" length="2"/>
<text>CD</text>
</annotation>
<annotation id="190">
<infon key="score">0.99923486</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
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<location offset="8229" length="14"/>
<text>Ig-like module</text>
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<annotation id="191">
<infon key="score">0.9880321</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8248" length="5"/>
<text>CBM46</text>
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<annotation id="192">
<infon key="score">0.99734473</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8261" length="15"/>
<text>β-sandwich fold</text>
</annotation>
<annotation id="193">
<infon key="score">0.89907354</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8293" length="8"/>
<text>β-sheets</text>
</annotation>
<annotation id="194">
<infon key="score">0.99596715</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8320" length="22"/>
<text>antiparallel β-strands</text>
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<annotation id="195">
<infon key="score">0.9775023</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8361" length="5"/>
<text>loops</text>
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<annotation id="823">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:14Z</infon>
<location offset="8379" length="7"/>
<text>α helix</text>
</annotation>
<annotation id="196">
<infon key="score">0.96771604</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8395" length="7"/>
<text>strands</text>
</annotation>
<annotation id="197">
<infon key="score">0.99835396</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8403" length="2"/>
<text>β3</text>
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<annotation id="198">
<infon key="score">0.9979388</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:57:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8410" length="2"/>
<text>β4</text>
</annotation>
<annotation id="199">
<infon key="score">0.9986167</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8426" length="21"/>
<text>structural comparison</text>
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<annotation id="200">
<infon key="score">0.99914145</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8460" length="14"/>
<text>Ig-like module</text>
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<annotation id="201">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8483" length="5"/>
<text>CBM46</text>
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<annotation id="202">
<infon key="score">0.9988082</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8499" length="11"/>
<text>Dali server</text>
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<annotation id="203">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8522" length="4"/>
<text>rmsd</text>
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<annotation id="204">
<infon key="score">0.99829483</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8542" length="7"/>
<text>Z-score</text>
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<annotation id="205">
<infon key="score">0.9988423</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8656" length="22"/>
<text>structure-based search</text>
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<annotation id="206">
<infon key="score">0.99914235</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8727" length="14"/>
<text>Ig-like module</text>
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<annotation id="207">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8760" length="14"/>
<text>Ig-like module</text>
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<annotation id="894">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:55Z</infon>
<location offset="8791" length="6"/>
<text>solved</text>
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<annotation id="208">
<infon key="score">0.99865067</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8798" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="209">
<infon key="score">0.85789466</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8821" length="11"/>
<text>tri-modular</text>
</annotation>
<annotation id="812">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="8833" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="210">
<infon key="score">0.99749374</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8851" length="19"/>
<text>Bacillus halodurans</text>
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<annotation id="211">
<infon key="score">0.990575</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8872" length="7"/>
<text>BhCel5B</text>
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<annotation id="212">
<infon key="score">0.9986528</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8886" length="4"/>
<text>rmsd</text>
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<annotation id="213">
<infon key="score">0.9982287</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8903" length="7"/>
<text>Z-score</text>
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<annotation id="214">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8923" length="5"/>
<text>CBM46</text>
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<annotation id="215">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8934" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="216">
<infon key="score">0.99646795</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8978" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="217">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8986" length="5"/>
<text>CBM46</text>
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<annotation id="218">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8998" length="4"/>
<text>rmsd</text>
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<annotation id="219">
<infon key="score">0.99820775</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9015" length="7"/>
<text>Z-score</text>
</annotation>
<annotation id="220">
<infon key="score">0.9766565</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9065" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="221">
<infon key="score">0.9958629</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9099" length="7"/>
<text>Ig-like</text>
</annotation>
<annotation id="222">
<infon key="score">0.9557978</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9119" length="5"/>
<text>CBM46</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9161</offset>
<text>The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues. Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module. In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.</text>
<annotation id="223">
<infon key="score">0.9992533</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9165" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="224">
<infon key="score">0.99951124</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9197" length="2"/>
<text>CD</text>
</annotation>
<annotation id="225">
<infon key="score">0.99710685</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9219" length="8"/>
<text>tyrosine</text>
</annotation>
<annotation id="226">
<infon key="score">0.9993705</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9229" length="4"/>
<text>Y367</text>
</annotation>
<annotation id="227">
<infon key="score">0.9975345</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9261" length="10"/>
<text>tryptophan</text>
</annotation>
<annotation id="228">
<infon key="score">0.9978206</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9329" length="7"/>
<text>glucose</text>
</annotation>
<annotation id="229">
<infon key="score">0.9991925</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9434" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="230">
<infon key="score">0.991456</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9467" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="231">
<infon key="score">0.9973424</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9483" length="10"/>
<text>tryptophan</text>
</annotation>
<annotation id="232">
<infon key="score">0.9994098</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9526" length="2"/>
<text>CD</text>
</annotation>
<annotation id="233">
<infon key="score">0.9986832</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9529" length="22"/>
<text>substrate binding site</text>
</annotation>
<annotation id="234">
<infon key="score">0.9937337</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9613" length="12"/>
<text>carbohydrate</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9635</offset>
<text>Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule. The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end. The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A). The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D). These residues belong to the CD and are conserved in the GH5 family.</text>
<annotation id="235">
<infon key="score">0.9983968</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9635" length="21"/>
<text>Electron density maps</text>
</annotation>
<annotation id="827">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:00Z</infon>
<location offset="9676" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="236">
<infon key="score">0.9993093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:13Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9690" length="13"/>
<text>cellotetraose</text>
</annotation>
<annotation id="237">
<infon key="score">0.803788</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9705" length="2"/>
<text>C4</text>
</annotation>
<annotation id="238">
<infon key="score">0.99934393</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9726" length="13"/>
<text>cellopentaose</text>
</annotation>
<annotation id="239">
<infon key="score">0.8238797</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9741" length="2"/>
<text>C5</text>
</annotation>
<annotation id="240">
<infon key="score">0.99800676</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9752" length="2"/>
<text>CD</text>
</annotation>
<annotation id="241">
<infon key="score">0.99895775</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9755" length="35"/>
<text>negative substrate-binding subsites</text>
</annotation>
<annotation id="242">
<infon key="score">0.99930835</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9818" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="243">
<infon key="score">0.99286747</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9829" length="20"/>
<text>catalytically active</text>
</annotation>
<annotation id="244">
<infon key="score">0.6759651</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9892" length="2"/>
<text>C5</text>
</annotation>
<annotation id="825">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:00:23Z</infon>
<location offset="9909" length="24"/>
<text>lack of electron density</text>
</annotation>
<annotation id="245">
<infon key="score">0.9951534</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9947" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="246">
<infon key="score">0.94431627</infon>
<infon key="type">residue_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:00:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9962" length="5"/>
<text>fifth</text>
</annotation>
<annotation id="247">
<infon key="score">0.99872655</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9968" length="7"/>
<text>glucose</text>
</annotation>
<annotation id="248">
<infon key="score">0.8547157</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9999" length="2"/>
<text>C5</text>
</annotation>
<annotation id="249">
<infon key="score">0.99846256</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10034" length="9"/>
<text>structure</text>
</annotation>
<annotation id="826">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:00:59Z</infon>
<location offset="10063" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="250">
<infon key="score">0.9401454</infon>
<infon key="type">residue_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:00:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10077" length="5"/>
<text>fifth</text>
</annotation>
<annotation id="251">
<infon key="score">0.99892324</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10083" length="7"/>
<text>glucose</text>
</annotation>
<annotation id="252">
<infon key="score">0.9905514</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10132" length="18"/>
<text>catalytic residues</text>
</annotation>
<annotation id="253">
<infon key="score">0.999263</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10186" length="4"/>
<text>R234</text>
</annotation>
<annotation id="254">
<infon key="score">0.9992963</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10269" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="255">
<infon key="score">0.95710206</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10287" length="2"/>
<text>C5</text>
</annotation>
<annotation id="256">
<infon key="score">0.9987225</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10295" length="7"/>
<text>glucose</text>
</annotation>
<annotation id="257">
<infon key="score">0.9217632</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10307" length="2"/>
<text>C4</text>
</annotation>
<annotation id="258">
<infon key="score">0.9981227</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:50:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10352" length="43"/>
<text>enzymatic product profile mass spectrometry</text>
</annotation>
<annotation id="259">
<infon key="score">0.4754696</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10420" length="2"/>
<text>C4</text>
</annotation>
<annotation id="794">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<location offset="10439" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="831">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:49Z</infon>
<location offset="10447" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="260">
<infon key="score">0.9821175</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10463" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="261">
<infon key="score">0.9969641</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10478" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="262">
<infon key="score">0.9992698</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10505" length="3"/>
<text>N36</text>
</annotation>
<annotation id="263">
<infon key="score">0.99924517</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10510" length="4"/>
<text>H113</text>
</annotation>
<annotation id="264">
<infon key="score">0.999233</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10516" length="4"/>
<text>H114</text>
</annotation>
<annotation id="265">
<infon key="score">0.9993267</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10522" length="4"/>
<text>N158</text>
</annotation>
<annotation id="266">
<infon key="score">0.9993518</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10528" length="4"/>
<text>W301</text>
</annotation>
<annotation id="267">
<infon key="score">0.9993304</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10538" length="4"/>
<text>N303</text>
</annotation>
<annotation id="833">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:54Z</infon>
<location offset="10552" length="16"/>
<text>CH-π interaction</text>
</annotation>
<annotation id="268">
<infon key="score">0.99936026</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:02:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10582" length="3"/>
<text>W47</text>
</annotation>
<annotation id="269">
<infon key="score">0.9988709</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10626" length="2"/>
<text>CD</text>
</annotation>
<annotation id="270">
<infon key="score">0.99867034</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10637" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="805">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<location offset="10654" length="3"/>
<text>GH5</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>10667</offset>
<text>BlCel5B enzymatic activity</text>
<annotation id="271">
<infon key="score">0.9967526</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10667" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10694</offset>
<text>BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C). BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel. Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D). Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.</text>
<annotation id="272">
<infon key="score">0.99664414</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10694" length="7"/>
<text>BlCel5B</text>
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<annotation id="273">
<infon key="score">0.9986595</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10735" length="22"/>
<text>carboxymethylcellulose</text>
</annotation>
<annotation id="274">
<infon key="score">0.99820554</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10759" length="3"/>
<text>CMC</text>
</annotation>
<annotation id="275">
<infon key="score">0.99701774</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10922" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="800">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<location offset="10938" length="6"/>
<text>active</text>
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<annotation id="276">
<infon key="score">0.9989734</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:51:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10948" length="8"/>
<text>β-glucan</text>
</annotation>
<annotation id="277">
<infon key="score">0.998659</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10968" length="8"/>
<text>lichenan</text>
</annotation>
<annotation id="278">
<infon key="score">0.99895394</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10993" length="10"/>
<text>xyloglucan</text>
</annotation>
<annotation id="279">
<infon key="score">0.9986243</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11074" length="13"/>
<text>galactomannan</text>
</annotation>
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<infon key="score">0.9518401</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11089" length="3"/>
<text>rye</text>
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<annotation id="281">
<infon key="score">0.99827766</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11093" length="12"/>
<text>arabinoxylan</text>
</annotation>
<annotation id="282">
<infon key="score">0.99909824</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:07Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11107" length="12"/>
<text>1,4-β-mannan</text>
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<annotation id="283">
<infon key="score">0.9968621</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11147" length="10"/>
<text>Azo-Avicel</text>
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<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:39Z</infon>
<location offset="11203" length="25"/>
<text>Michaelis-Menten behavior</text>
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<annotation id="284">
<infon key="score">0.99784744</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11234" length="3"/>
<text>CMC</text>
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<annotation id="285">
<infon key="score">0.9709084</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11252" length="2"/>
<text>KM</text>
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<annotation id="286">
<infon key="score">0.98771733</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:04:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11272" length="4"/>
<text>Vmax</text>
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<infon key="score">0.99737585</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11330" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="801">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<location offset="11354" length="6"/>
<text>active</text>
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<annotation id="813">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="11421" length="5"/>
<text>GH5_4</text>
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<annotation id="802">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<location offset="11458" length="6"/>
<text>active</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>11580</offset>
<text>To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A. These mutants were expressed and purified as described for the wild-type enzyme. Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity. Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1). All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:47Z</infon>
<location offset="11616" length="17"/>
<text>ancillary modules</text>
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<annotation id="288">
<infon key="score">0.99895215</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11638" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="289">
<infon key="score">0.99880445</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11656" length="16"/>
<text>enzymatic assays</text>
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<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:23Z</infon>
<location offset="11708" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="290">
<infon key="score">0.9084131</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11719" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="291">
<infon key="score">0.5755105</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11725" length="8"/>
<text>deletion</text>
</annotation>
<annotation id="292">
<infon key="score">0.9981463</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11735" length="6"/>
<text>ΔCBM46</text>
</annotation>
<annotation id="837">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:35Z</infon>
<location offset="11750" length="7"/>
<text>Ig-like</text>
</annotation>
<annotation id="293">
<infon key="score">0.97647095</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11760" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="294">
<infon key="score">0.7081323</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11766" length="8"/>
<text>deletion</text>
</annotation>
<annotation id="295">
<infon key="score">0.9988201</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11776" length="9"/>
<text>ΔIg-CBM46</text>
</annotation>
<annotation id="296">
<infon key="score">0.99609196</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11798" length="15"/>
<text>point mutations</text>
</annotation>
<annotation id="297">
<infon key="score">0.3921337</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11821" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="298">
<infon key="score">0.9528272</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11850" length="5"/>
<text>W479A</text>
</annotation>
<annotation id="299">
<infon key="score">0.9781924</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11860" length="5"/>
<text>W481A</text>
</annotation>
<annotation id="840">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:23Z</infon>
<location offset="11873" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="300">
<infon key="score">0.99014854</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11886" length="22"/>
<text>expressed and purified</text>
</annotation>
<annotation id="301">
<infon key="score">0.99896145</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11930" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="841">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:46Z</infon>
<location offset="11975" length="17"/>
<text>deletion variants</text>
</annotation>
<annotation id="302">
<infon key="score">0.9990125</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12065" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="303">
<infon key="score">0.9991658</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12077" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="304">
<infon key="score">0.99874085</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12119" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="305">
<infon key="score">0.94216156</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12142" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="306">
<infon key="score">0.9989291</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12166" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="307">
<infon key="score">0.9990215</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12184" length="20"/>
<text>Thermal shift assays</text>
</annotation>
<annotation id="308">
<infon key="score">0.70613146</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12259" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="309">
<infon key="score">0.99822545</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:05:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12328" length="20"/>
<text>melting temperatures</text>
</annotation>
<annotation id="310">
<infon key="score">0.9989581</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12360" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="311">
<infon key="score">0.9986719</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12379" length="13"/>
<text>BlCel5BΔCBM46</text>
</annotation>
<annotation id="312">
<infon key="score">0.9987348</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12404" length="16"/>
<text>BlCel5BΔIg-CBM46</text>
</annotation>
<annotation id="313">
<infon key="score">0.99815816</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12432" length="12"/>
<text>BlCel5BW479A</text>
</annotation>
<annotation id="314">
<infon key="score">0.9983828</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12459" length="12"/>
<text>BlCel5BW479A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12539</offset>
<text>We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants. Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC). This indicates that CBM46 must interact with the substrate via residues W479 and W481. However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.</text>
<annotation id="315">
<infon key="score">0.99529123</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12576" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="316">
<infon key="score">0.5860964</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12588" length="7"/>
<text>surface</text>
</annotation>
<annotation id="317">
<infon key="score">0.9995022</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12605" length="4"/>
<text>W479</text>
</annotation>
<annotation id="318">
<infon key="score">0.9995104</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12614" length="4"/>
<text>W481</text>
</annotation>
<annotation id="319">
<infon key="score">0.9991447</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12632" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="320">
<infon key="score">0.99821043</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12663" length="16"/>
<text>enzymatic assays</text>
</annotation>
<annotation id="321">
<infon key="score">0.9988212</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12685" length="5"/>
<text>W479A</text>
</annotation>
<annotation id="322">
<infon key="score">0.998841</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12695" length="5"/>
<text>W481A</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:07:23Z</infon>
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<location offset="12701" length="7"/>
<text>mutants</text>
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<infon key="updated_at">2023-09-21T10:08:37Z</infon>
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<location offset="12715" length="9"/>
<text>mutations</text>
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<location offset="12797" length="5"/>
<text>W481A</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:59Z</infon>
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<location offset="12833" length="5"/>
<text>W479A</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12874" length="2"/>
<text>wt</text>
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<annotation id="328">
<infon key="score">0.99914324</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
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<location offset="12877" length="7"/>
<text>BlCel5B</text>
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<infon key="updated_at">2023-09-21T09:51:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12891" length="8"/>
<text>β-glucan</text>
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<infon key="updated_at">2023-09-21T10:03:31Z</infon>
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<text>CBM46</text>
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<infon key="updated_at">2023-09-21T10:08:25Z</infon>
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<text>W479</text>
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<infon key="updated_at">2023-09-21T10:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13010" length="4"/>
<text>W481</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13035" length="7"/>
<text>BlCel5B</text>
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<annotation id="335">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13043" length="17"/>
<text>crystal structure</text>
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<text>close</text>
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<text>CBM46</text>
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<infon key="updated_at">2023-09-21T09:44:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13262" length="12"/>
<text>carbohydrate</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
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<text>BlCelB5 dynamics and binding-site architecture</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:48:04Z</infon>
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<location offset="13279" length="7"/>
<text>BlCelB5</text>
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<annotation id="339">
<infon key="score">0.998885</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13300" length="12"/>
<text>binding-site</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13326</offset>
<text>Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B. In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A). This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product. No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B). However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).</text>
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<infon key="updated_at">2023-09-21T10:09:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13326" length="18"/>
<text>Molecular dynamics</text>
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<infon key="updated_at">2023-09-21T10:09:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13346" length="2"/>
<text>MD</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13350" length="11"/>
<text>simulations</text>
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<annotation id="343">
<infon key="score">0.99915814</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13423" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="344">
<infon key="score">0.9986778</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13439" length="11"/>
<text>simulations</text>
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<annotation id="345">
<infon key="score">0.9987263</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13458" length="17"/>
<text>crystal structure</text>
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<annotation id="346">
<infon key="score">0.99925226</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13480" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="842">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:34Z</infon>
<location offset="13488" length="8"/>
<text>bound to</text>
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<annotation id="830">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:09Z</infon>
<location offset="13497" length="2"/>
<text>C4</text>
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<annotation id="347">
<infon key="score">0.99466866</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13575" length="10"/>
<text>simulation</text>
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<annotation id="348">
<infon key="score">0.9992524</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13647" length="13"/>
<text>cellotetraose</text>
</annotation>
<annotation id="349">
<infon key="score">0.9991716</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13716" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="350">
<infon key="score">0.99924564</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13875" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="351">
<infon key="score">0.9994252</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13973" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="352">
<infon key="score">0.9992703</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13983" length="15"/>
<text>Ig-like domains</text>
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<annotation id="353">
<infon key="score">0.9994916</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14051" length="2"/>
<text>CD</text>
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<annotation id="354">
<infon key="score">0.99847704</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:54:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14060" length="4"/>
<text>rmsd</text>
</annotation>
<annotation id="355">
<infon key="score">0.9992304</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14126" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>14243</offset>
<text>Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions. aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima. aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated. During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1). Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate. Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively. Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations. During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site. These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.</text>
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<infon key="score">0.9952859</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14256" length="11"/>
<text>simulations</text>
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<infon key="score">0.9983346</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14294" length="30"/>
<text>accelerated molecular dynamics</text>
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<annotation id="358">
<infon key="score">0.99809784</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14326" length="3"/>
<text>aMD</text>
</annotation>
<annotation id="359">
<infon key="score">0.9991818</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14351" length="7"/>
<text>BlCel5B</text>
</annotation>
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<infon key="score">0.99624324</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14380" length="3"/>
<text>aMD</text>
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<annotation id="361">
<infon key="score">0.9771267</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14446" length="33"/>
<text>dihedral potential energy surface</text>
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<annotation id="362">
<infon key="score">0.9982533</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14611" length="3"/>
<text>aMD</text>
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<annotation id="363">
<infon key="score">0.98539543</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14615" length="12"/>
<text>trajectories</text>
</annotation>
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<infon key="score">0.997635</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14678" length="2"/>
<text>MD</text>
</annotation>
<annotation id="365">
<infon key="score">0.99686617</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14715" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="366">
<infon key="score">0.9858672</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14772" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="367">
<infon key="score">0.99897504</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14782" length="2"/>
<text>CD</text>
</annotation>
<annotation id="368">
<infon key="score">0.99891853</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14858" length="11"/>
<text>active site</text>
</annotation>
<annotation id="369">
<infon key="score">0.99922407</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14898" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="370">
<infon key="score">0.982692</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14913" length="16"/>
<text>crystallographic</text>
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<annotation id="371">
<infon key="score">0.9982843</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14995" length="3"/>
<text>aMD</text>
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<annotation id="372">
<infon key="score">0.9991255</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15013" length="10"/>
<text>absence of</text>
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<annotation id="844">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:10Z</infon>
<location offset="15100" length="8"/>
<text>distance</text>
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<annotation id="373">
<infon key="score">0.99943405</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15143" length="4"/>
<text>I120</text>
</annotation>
<annotation id="374">
<infon key="score">0.999323</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15152" length="4"/>
<text>E477</text>
</annotation>
<annotation id="375">
<infon key="score">0.9990532</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15211" length="2"/>
<text>CD</text>
</annotation>
<annotation id="376">
<infon key="score">0.9609153</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15218" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="377">
<infon key="score">0.99820685</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15264" length="4"/>
<text>I120</text>
</annotation>
<annotation id="378">
<infon key="score">0.9978446</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15269" length="4"/>
<text>E477</text>
</annotation>
<annotation id="379">
<infon key="score">0.59275645</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15274" length="8"/>
<text>distance</text>
</annotation>
<annotation id="380">
<infon key="score">0.99833626</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15370" length="3"/>
<text>aMD</text>
</annotation>
<annotation id="381">
<infon key="score">0.9909917</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15374" length="10"/>
<text>trajectory</text>
</annotation>
<annotation id="382">
<infon key="score">0.9992021</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15422" length="9"/>
<text>semi-open</text>
</annotation>
<annotation id="383">
<infon key="score">0.6727852</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15433" length="16"/>
<text>crystallographic</text>
</annotation>
<annotation id="384">
<infon key="score">0.9992982</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15455" length="8"/>
<text>occluded</text>
</annotation>
<annotation id="843">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:02Z</infon>
<location offset="15465" length="3"/>
<text>aMD</text>
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<annotation id="850">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:52Z</infon>
<location offset="15524" length="14"/>
<text>aMD simulation</text>
</annotation>
<annotation id="385">
<infon key="score">0.99914116</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15544" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="386">
<infon key="score">0.9993155</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15579" length="6"/>
<text>closed</text>
</annotation>
<annotation id="387">
<infon key="score">0.86605585</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15609" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="388">
<infon key="score">0.99907833</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15628" length="25"/>
<text>carbohydrate-binding site</text>
</annotation>
<annotation id="389">
<infon key="score">0.99925894</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15682" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="390">
<infon key="score">0.38964275</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15767" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="391">
<infon key="score">0.9977906</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15791" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="392">
<infon key="score">0.9989761</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15804" length="2"/>
<text>CD</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15823</offset>
<text>To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme. Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D). This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E). However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B). In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C). This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).</text>
<annotation id="393">
<infon key="score">0.9991779</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15852" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="394">
<infon key="score">0.9978961</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:53:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15882" length="6"/>
<text>glucan</text>
</annotation>
<annotation id="395">
<infon key="score">0.61043835</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15913" length="9"/>
<text>structure</text>
</annotation>
<annotation id="396">
<infon key="score">0.9992067</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15930" length="12"/>
<text>cellooctaose</text>
</annotation>
<annotation id="397">
<infon key="score">0.9925861</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15944" length="2"/>
<text>C8</text>
</annotation>
<annotation id="895">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:52:17Z</infon>
<location offset="15974" length="19"/>
<text>positive (+1 to +4)</text>
</annotation>
<annotation id="896">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:52:36Z</infon>
<location offset="15998" length="19"/>
<text>negative (−4 to −1)</text>
</annotation>
<annotation id="398">
<infon key="score">0.9882339</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:52:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16018" length="8"/>
<text>subsites</text>
</annotation>
<annotation id="399">
<infon key="score">0.99916506</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16077" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="400">
<infon key="score">0.89470834</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16103" length="2"/>
<text>C8</text>
</annotation>
<annotation id="401">
<infon key="score">0.9989809</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16147" length="11"/>
<text>active site</text>
</annotation>
<annotation id="402">
<infon key="score">0.99927276</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16261" length="4"/>
<text>open</text>
</annotation>
<annotation id="403">
<infon key="score">0.99922884</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16282" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="404">
<infon key="score">0.9962482</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:06Z</infon>
<infon key="identifier">GO:</infon>
<location offset="16417" length="10"/>
<text>BlCel5B-C8</text>
</annotation>
<annotation id="405">
<infon key="score">0.8329121</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16448" length="14"/>
<text>aMD simulation</text>
</annotation>
<annotation id="406">
<infon key="score">0.923577</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16495" length="2"/>
<text>C8</text>
</annotation>
<annotation id="407">
<infon key="score">0.901596</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16571" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="408">
<infon key="score">0.98974496</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16585" length="6"/>
<text>closed</text>
</annotation>
<annotation id="409">
<infon key="score">0.99818546</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16601" length="2"/>
<text>CD</text>
</annotation>
<annotation id="410">
<infon key="score">0.9084791</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16620" length="2"/>
<text>C8</text>
</annotation>
<annotation id="828">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:00Z</infon>
<location offset="16674" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="411">
<infon key="score">0.6347472</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16701" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="851">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:43Z</infon>
<location offset="16760" length="26"/>
<text>crystallographic structure</text>
</annotation>
<annotation id="412">
<infon key="score">0.9988453</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16812" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="413">
<infon key="score">0.9980185</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16827" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="414">
<infon key="score">0.99516326</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16835" length="15"/>
<text>aMD simulations</text>
</annotation>
<annotation id="415">
<infon key="score">0.99944776</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16879" length="4"/>
<text>I120</text>
</annotation>
<annotation id="416">
<infon key="score">0.99935466</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16884" length="4"/>
<text>E477</text>
</annotation>
<annotation id="845">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<location offset="16889" length="8"/>
<text>distance</text>
</annotation>
<annotation id="417">
<infon key="score">0.99933016</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16933" length="6"/>
<text>closed</text>
</annotation>
<annotation id="418">
<infon key="score">0.8814976</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16969" length="2"/>
<text>C8</text>
</annotation>
<annotation id="419">
<infon key="score">0.9988757</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17006" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="420">
<infon key="score">0.9991295</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17021" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="421">
<infon key="score">0.9085049</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:07Z</infon>
<infon key="identifier">GO:</infon>
<location offset="17046" length="10"/>
<text>BlCel5B-C8</text>
</annotation>
<annotation id="854">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:05Z</infon>
<location offset="17128" length="13"/>
<text>MD simulation</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17220</offset>
<text>A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D. Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.</text>
<annotation id="422">
<infon key="score">0.99593264</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17301" length="11"/>
<text>simulations</text>
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<annotation id="423">
<infon key="score">0.98428255</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17330" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="424">
<infon key="score">0.99671966</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17336" length="10"/>
<text>tryptophan</text>
</annotation>
<annotation id="425">
<infon key="score">0.99955076</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17356" length="4"/>
<text>W479</text>
</annotation>
<annotation id="426">
<infon key="score">0.9995515</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17365" length="4"/>
<text>W481</text>
</annotation>
<annotation id="427">
<infon key="score">0.8669198</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17382" length="2"/>
<text>CD</text>
</annotation>
<annotation id="428">
<infon key="score">0.99655175</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17385" length="10"/>
<text>tryptophan</text>
</annotation>
<annotation id="429">
<infon key="score">0.9840515</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17430" length="12"/>
<text>carbohydrate</text>
</annotation>
<annotation id="897">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:52:54Z</infon>
<location offset="17485" length="6"/>
<text>tunnel</text>
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<annotation id="875">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:28:45Z</infon>
<location offset="17516" length="7"/>
<text>BlCel5B</text>
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<annotation id="876">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:28:55Z</infon>
<location offset="17524" length="13"/>
<text>binding cleft</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17601" length="5"/>
<text>CBM46</text>
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<annotation id="431">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17633" length="16"/>
<text>catalytic active</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
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<location offset="17694" length="5"/>
<text>CBM46</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17743" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17752</offset>
<text>To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations. We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling. The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale. The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.</text>
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<infon key="updated_at">2023-09-21T10:15:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17807" length="21"/>
<text>atomistic simulations</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17866" length="7"/>
<text>BlCel5B</text>
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<infon key="updated_at">2023-09-21T10:15:13Z</infon>
<infon key="identifier">MESH:</infon>
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<text>coarse-grained MD</text>
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<infon key="updated_at">2023-09-21T10:15:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17899" length="5"/>
<text>CG-MD</text>
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<annotation id="438">
<infon key="score">0.99404365</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17906" length="11"/>
<text>simulations</text>
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<annotation id="855">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:15:33Z</infon>
<location offset="17958" length="17"/>
<text>CG-MD simulations</text>
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<annotation id="439">
<infon key="score">0.97689587</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18030" length="8"/>
<text>distance</text>
</annotation>
<annotation id="440">
<infon key="score">0.99928975</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18105" length="2"/>
<text>CD</text>
</annotation>
<annotation id="441">
<infon key="score">0.99608094</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18112" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="442">
<infon key="score">0.9849777</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:15:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18274" length="30"/>
<text>computed distance distribution</text>
</annotation>
<annotation id="443">
<infon key="score">0.9993337</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18385" length="6"/>
<text>closed</text>
</annotation>
<annotation id="444">
<infon key="score">0.9988933</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18442" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="848">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:40Z</infon>
<location offset="18457" length="15"/>
<text>aMD simulations</text>
</annotation>
<annotation id="445">
<infon key="score">0.9874643</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18483" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="446">
<infon key="score">0.9985495</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18508" length="2"/>
<text>CD</text>
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<annotation id="447">
<infon key="score">0.99901456</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18524" length="22"/>
<text>substrate binding site</text>
</annotation>
<annotation id="448">
<infon key="score">0.99915606</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18556" length="9"/>
<text>semi-open</text>
</annotation>
<annotation id="852">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:43Z</infon>
<location offset="18595" length="26"/>
<text>crystallographic structure</text>
</annotation>
<annotation id="449">
<infon key="score">0.9993492</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18633" length="8"/>
<text>extended</text>
</annotation>
<annotation id="450">
<infon key="score">0.9991979</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18642" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="451">
<infon key="score">0.99903035</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18677" length="2"/>
<text>CD</text>
</annotation>
<annotation id="452">
<infon key="score">0.99208415</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18684" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="453">
<infon key="score">0.99749935</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18725" length="17"/>
<text>crystal structure</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>18751</offset>
<text>BlCel5B conformers fit the SAXS envelope</text>
<annotation id="454">
<infon key="score">0.9979134</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18751" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="455">
<infon key="score">0.9971818</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18778" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="456">
<infon key="score">0.9409911</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18783" length="8"/>
<text>envelope</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18792</offset>
<text>SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2). The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).</text>
<annotation id="457">
<infon key="score">0.99888974</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18792" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="458">
<infon key="score">0.99892706</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18834" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="459">
<infon key="score">0.9987596</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18920" length="9"/>
<text>monomeric</text>
</annotation>
<annotation id="460">
<infon key="score">0.9957338</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18959" length="2"/>
<text>Rg</text>
</annotation>
<annotation id="461">
<infon key="score">0.9926064</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18976" length="4"/>
<text>Dmax</text>
</annotation>
<annotation id="462">
<infon key="score">0.9984776</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19020" length="26"/>
<text>ab initio dummy atom model</text>
</annotation>
<annotation id="463">
<infon key="score">0.9986356</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19048" length="3"/>
<text>DAM</text>
</annotation>
<annotation id="464">
<infon key="score">0.99829227</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19075" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="465">
<infon key="score">0.9989692</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19088" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="856">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:19Z</infon>
<location offset="19106" length="8"/>
<text>envelope</text>
</annotation>
<annotation id="466">
<infon key="score">0.99870574</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19211" length="11"/>
<text>simulations</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>19248</offset>
<text>It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions. Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3). Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies. We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope. The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).</text>
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<infon key="score">0.99568725</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19267" length="11"/>
<text>Kratky plot</text>
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<annotation id="468">
<infon key="score">0.99910104</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19503" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="857">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:13Z</infon>
<location offset="19754" length="31"/>
<text>crystallographic and MD studies</text>
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<infon key="score">0.9935033</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19805" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="470">
<infon key="score">0.9407316</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19810" length="8"/>
<text>envelope</text>
</annotation>
<annotation id="471">
<infon key="score">0.998749</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19862" length="15"/>
<text>superimposition</text>
</annotation>
<annotation id="472">
<infon key="score">0.999127</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19939" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="473">
<infon key="score">0.99936813</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19960" length="6"/>
<text>closed</text>
</annotation>
<annotation id="872">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:41:06Z</infon>
<location offset="19970" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="873">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:41:16Z</infon>
<location offset="19976" length="2"/>
<text>CD</text>
</annotation>
<annotation id="874">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:41:26Z</infon>
<location offset="19979" length="8"/>
<text>occluded</text>
</annotation>
<annotation id="474">
<infon key="score">0.9985977</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20020" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="475">
<infon key="score">0.99917656</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20065" length="9"/>
<text>semi-open</text>
</annotation>
<annotation id="476">
<infon key="score">0.9984336</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20107" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="477">
<infon key="score">0.9993529</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20154" length="8"/>
<text>extended</text>
</annotation>
<annotation id="478">
<infon key="score">0.9984976</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20185" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="479">
<infon key="score">0.98378396</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20232" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="480">
<infon key="score">0.9104051</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20237" length="8"/>
<text>envelope</text>
</annotation>
<annotation id="481">
<infon key="score">0.9977047</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20261" length="24"/>
<text>average scattering curve</text>
</annotation>
<annotation id="482">
<infon key="score">0.99628973</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20332" length="20"/>
<text>scattering intensity</text>
</annotation>
<annotation id="483">
<infon key="score">0.994159</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20359" length="1"/>
<text>χ</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>20397</offset>
<text>GH5_4 phylogenetic analysis</text>
<annotation id="814">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="20397" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="484">
<infon key="score">0.99817145</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20403" length="21"/>
<text>phylogenetic analysis</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>20425</offset>
<text>To date, there are 427 sequences classified as subfamily 4 members in the CAZy database. After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A). According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B). Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region. Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor. These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.</text>
<annotation id="485">
<infon key="score">0.8797403</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20664" length="11"/>
<text>277 and 400</text>
</annotation>
<annotation id="486">
<infon key="score">0.99440295</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20690" length="7"/>
<text>aligned</text>
</annotation>
<annotation id="862">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:20:09Z</infon>
<location offset="20722" length="17"/>
<text>phylogenetic tree</text>
</annotation>
<annotation id="806">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<location offset="20829" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="487">
<infon key="score">0.99921066</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:19:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20890" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="860">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:19:29Z</infon>
<location offset="20910" length="6"/>
<text>CBM_X2</text>
</annotation>
<annotation id="488">
<infon key="score">0.99906105</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:19:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21059" length="4"/>
<text>CBM1</text>
</annotation>
<annotation id="489">
<infon key="score">0.9988931</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:19:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21078" length="4"/>
<text>CBM2</text>
</annotation>
<annotation id="490">
<infon key="score">0.99761593</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21207" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="815">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="21230" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="861">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:20:08Z</infon>
<location offset="21291" length="17"/>
<text>phylogenetic tree</text>
</annotation>
<annotation id="491">
<infon key="score">0.99684453</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21557" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="492">
<infon key="score">0.9991983</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21574" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="863">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:20:35Z</infon>
<location offset="21590" length="6"/>
<text>CBM_X2</text>
</annotation>
<annotation id="493">
<infon key="score">0.9989972</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:20:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21603" length="16"/>
<text>structural hinge</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>21621</offset>
<text>Discussion</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>21632</offset>
<text>Growing interest in biotechnological applications of enzymes exhibiting activity toward lignocellulosic biomass has sparked efforts in the discovery and development of novel enzymes, as well as the search for a deeper understanding of their mechanisms of action. Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity. Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.</text>
<annotation id="869">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:12Z</infon>
<location offset="21918" length="10"/>
<text>trimodular</text>
</annotation>
<annotation id="494">
<infon key="score">0.999111</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21959" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="495">
<infon key="score">0.99920964</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21971" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="816">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="21996" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="496">
<infon key="score">0.9991419</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22117" length="11"/>
<text>Full-length</text>
</annotation>
<annotation id="497">
<infon key="score">0.9992415</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22129" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="498">
<infon key="score">0.98844063</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22140" length="6"/>
<text>active</text>
</annotation>
<annotation id="865">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:21:10Z</infon>
<location offset="22155" length="10"/>
<text>cellulosic</text>
</annotation>
<annotation id="866">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:21:21Z</infon>
<location offset="22170" length="14"/>
<text>hemicellulosic</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>22248</offset>
<text>Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs. AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate. Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately. In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B. We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site. Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis. Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs. This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case. To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.</text>
<annotation id="499">
<infon key="score">0.99675375</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:21:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22253" length="27"/>
<text>carbohydrate-active enzymes</text>
</annotation>
<annotation id="500">
<infon key="score">0.99907744</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22310" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="501">
<infon key="score">0.5816129</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22360" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="502">
<infon key="score">0.7359264</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22365" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="503">
<infon key="score">0.95438325</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22378" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="504">
<infon key="score">0.9982529</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22404" length="13"/>
<text>carbohydrates</text>
</annotation>
<annotation id="505">
<infon key="score">0.9990488</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22443" length="17"/>
<text>catalytic domains</text>
</annotation>
<annotation id="506">
<infon key="score">0.9198811</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:50:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22495" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="507">
<infon key="score">0.9958831</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:43:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22552" length="7"/>
<text>linkers</text>
</annotation>
<annotation id="508">
<infon key="score">0.9984366</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22721" length="23"/>
<text>protein crystallography</text>
</annotation>
<annotation id="509">
<infon key="score">0.9988351</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22746" length="32"/>
<text>computational molecular dynamics</text>
</annotation>
<annotation id="510">
<infon key="score">0.998632</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22784" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="511">
<infon key="score">0.999297</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22899" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="512">
<infon key="score">0.99921644</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22903" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="513">
<infon key="score">0.9176079</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22928" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="514">
<infon key="score">0.9990823</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22935" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="515">
<infon key="score">0.9992482</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22947" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="516">
<infon key="score">0.99905556</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22977" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="517">
<infon key="score">0.9524748</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22992" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="518">
<infon key="score">0.99903226</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23060" length="22"/>
<text>substrate-binding site</text>
</annotation>
<annotation id="519">
<infon key="score">0.9991215</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23097" length="2"/>
<text>CD</text>
</annotation>
<annotation id="520">
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23100" length="5"/>
<text>alone</text>
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<annotation id="521">
<infon key="score">0.8547185</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23162" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="522">
<infon key="score">0.6654872</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23174" length="4"/>
<text>open</text>
</annotation>
<annotation id="867">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:21:53Z</infon>
<location offset="23179" length="5"/>
<text>close</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23322" length="3"/>
<text>AMs</text>
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<annotation id="524">
<infon key="score">0.99891365</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23373" length="6"/>
<text>active</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23425" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="859">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<location offset="23489" length="8"/>
<text>extended</text>
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<annotation id="526">
<infon key="score">0.99912506</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23691" length="2"/>
<text>GH</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>23695</offset>
<text>The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46. In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5). Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs. Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning. Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism. The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6). This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6). Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates. A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering). Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.</text>
<annotation id="527">
<infon key="score">0.9990599</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23699" length="15"/>
<text>CD binding site</text>
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<annotation id="528">
<infon key="score">0.9987381</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23718" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="529">
<infon key="score">0.81928617</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23863" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="530">
<infon key="score">0.9991128</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:45:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23889" length="4"/>
<text>GH5s</text>
</annotation>
<annotation id="531">
<infon key="score">0.99896187</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23966" length="13"/>
<text>binding cleft</text>
</annotation>
<annotation id="532">
<infon key="score">0.99738413</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:52:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23983" length="6"/>
<text>tunnel</text>
</annotation>
<annotation id="533">
<infon key="score">0.99770784</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24131" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="534">
<infon key="score">0.9743686</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24148" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="535">
<infon key="score">0.99888396</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24175" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="536">
<infon key="score">0.99887174</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24191" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="537">
<infon key="score">0.99892867</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24218" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="807">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<location offset="24257" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="538">
<infon key="score">0.99877423</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24274" length="4"/>
<text>lack</text>
</annotation>
<annotation id="539">
<infon key="score">0.95958143</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24279" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="540">
<infon key="score">0.9986505</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24290" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="541">
<infon key="score">0.7155743</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24336" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="542">
<infon key="score">0.9988024</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24412" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="543">
<infon key="score">0.9987122</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24456" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="544">
<infon key="score">0.9529958</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24532" length="9"/>
<text>structure</text>
</annotation>
<annotation id="545">
<infon key="score">0.9982799</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24545" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="546">
<infon key="score">0.9981337</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24557" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="547">
<infon key="score">0.9990161</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24608" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="548">
<infon key="score">0.99565804</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24697" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="549">
<infon key="score">0.9989014</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24705" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="550">
<infon key="score">0.97640747</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24721" length="8"/>
<text>V-shaped</text>
</annotation>
<annotation id="551">
<infon key="score">0.99807286</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24750" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="552">
<infon key="score">0.9989003</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24758" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="553">
<infon key="score">0.99921954</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24806" length="4"/>
<text>loop</text>
</annotation>
<annotation id="554">
<infon key="score">0.99956995</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24828" length="4"/>
<text>F177</text>
</annotation>
<annotation id="555">
<infon key="score">0.9995683</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24837" length="4"/>
<text>R185</text>
</annotation>
<annotation id="556">
<infon key="score">0.9985153</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24847" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="557">
<infon key="score">0.9065534</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24856" length="6"/>
<text>absent</text>
</annotation>
<annotation id="558">
<infon key="score">0.99850297</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24870" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="559">
<infon key="score">0.9995596</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24903" length="4"/>
<text>W181</text>
</annotation>
<annotation id="560">
<infon key="score">0.9990684</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24931" length="13"/>
<text>binding cleft</text>
</annotation>
<annotation id="561">
<infon key="score">0.9984578</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24978" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="562">
<infon key="score">0.99243915</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24986" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="563">
<infon key="score">0.9990066</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25009" length="16"/>
<text>β-1,3-1,4-glucan</text>
</annotation>
<annotation id="564">
<infon key="score">0.99781764</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25038" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="803">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<location offset="25068" length="6"/>
<text>active</text>
</annotation>
<annotation id="565">
<infon key="score">0.99545187</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25075" length="7"/>
<text>without</text>
</annotation>
<annotation id="566">
<infon key="score">0.89027363</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25083" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="567">
<infon key="score">0.99849916</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25095" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="568">
<infon key="score">0.6794937</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25126" length="6"/>
<text>active</text>
</annotation>
<annotation id="569">
<infon key="score">0.99904317</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:49:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25141" length="10"/>
<text>xyloglucan</text>
</annotation>
<annotation id="570">
<infon key="score">0.9831435</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25173" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="571">
<infon key="score">0.9978708</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:18:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25285" length="21"/>
<text>phylogenetic analysis</text>
</annotation>
<annotation id="817">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="25351" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="572">
<infon key="score">0.97137105</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25370" length="10"/>
<text>trimodular</text>
</annotation>
<annotation id="573">
<infon key="score">0.9991372</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25492" length="4"/>
<text>loop</text>
</annotation>
<annotation id="574">
<infon key="score">0.9713052</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25522" length="11"/>
<text>161 and 163</text>
</annotation>
<annotation id="575">
<infon key="score">0.9986671</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25535" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="576">
<infon key="score">0.9987871</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25596" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="577">
<infon key="score">0.98740965</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25679" length="3"/>
<text>AMs</text>
</annotation>
<annotation id="578">
<infon key="score">0.9983164</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:42:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25701" length="5"/>
<text>plant</text>
</annotation>
<annotation id="579">
<infon key="score">0.9876605</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:37:18Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25717" length="15"/>
<text>polysaccharides</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>25816</offset>
<text>The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function. Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site. Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.</text>
<annotation id="580">
<infon key="score">0.9989795</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25847" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="581">
<infon key="score">0.99937105</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25855" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="582">
<infon key="score">0.99841034</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:51:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25887" length="6"/>
<text>BLASTP</text>
</annotation>
<annotation id="864">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:20:35Z</infon>
<location offset="25910" length="6"/>
<text>CBM_X2</text>
</annotation>
<annotation id="870">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:38:57Z</infon>
<location offset="25971" length="26"/>
<text>CBM-like accessory modules</text>
</annotation>
<annotation id="583">
<infon key="score">0.999084</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26052" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="584">
<infon key="score">0.99936295</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26060" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="585">
<infon key="score">0.9975055</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26078" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="586">
<infon key="score">0.99744666</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26131" length="25"/>
<text>carbohydrate-binding site</text>
</annotation>
<annotation id="587">
<infon key="score">0.99939424</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26201" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="588">
<infon key="score">0.9986139</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:38:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26257" length="16"/>
<text>structural hinge</text>
</annotation>
<annotation id="589">
<infon key="score">0.89083683</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26299" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="590">
<infon key="score">0.9985557</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26309" length="2"/>
<text>CD</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>26370</offset>
<text>Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture. BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7). In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46. After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release. The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme. These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process. Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis. SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.</text>
<annotation id="591">
<infon key="score">0.99876994</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26398" length="37"/>
<text>crystallographic, computer simulation</text>
</annotation>
<annotation id="592">
<infon key="score">0.91871595</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26441" length="24"/>
<text>SAXS structural analyses</text>
</annotation>
<annotation id="593">
<infon key="score">0.99891764</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26478" length="25"/>
<text>site-directed mutagenesis</text>
</annotation>
<annotation id="594">
<infon key="score">0.9507582</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26508" length="15"/>
<text>activity assays</text>
</annotation>
<annotation id="595">
<infon key="score">0.9991353</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26562" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="818">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="26616" length="5"/>
<text>GH5_4</text>
</annotation>
<annotation id="871">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:32Z</infon>
<location offset="26656" length="11"/>
<text>tri-modular</text>
</annotation>
<annotation id="596">
<infon key="score">0.99914575</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26682" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="597">
<infon key="score">0.9973775</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26759" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="598">
<infon key="score">0.9993305</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26765" length="2"/>
<text>CD</text>
</annotation>
<annotation id="599">
<infon key="score">0.9993292</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26768" length="6"/>
<text>closed</text>
</annotation>
<annotation id="600">
<infon key="score">0.9993451</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26779" length="8"/>
<text>occluded</text>
</annotation>
<annotation id="601">
<infon key="score">0.9993074</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26792" length="8"/>
<text>extended</text>
</annotation>
<annotation id="602">
<infon key="score">0.99929094</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26828" length="8"/>
<text>extended</text>
</annotation>
<annotation id="603">
<infon key="score">0.9987299</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26891" length="22"/>
<text>substrate binding site</text>
</annotation>
<annotation id="604">
<infon key="score">0.99939</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26917" length="2"/>
<text>CD</text>
</annotation>
<annotation id="605">
<infon key="score">0.9991791</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:39:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26934" length="11"/>
<text>semi-closed</text>
</annotation>
<annotation id="606">
<infon key="score">0.8726911</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27093" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="607">
<infon key="score">0.9992094</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27142" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="608">
<infon key="score">0.998947</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27192" length="11"/>
<text>active site</text>
</annotation>
<annotation id="609">
<infon key="score">0.99054337</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:40:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27271" length="41"/>
<text>mutagenesis and enzymatic activity assays</text>
</annotation>
<annotation id="610">
<infon key="score">0.99924946</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27334" length="14"/>
<text>Ig-like module</text>
</annotation>
<annotation id="611">
<infon key="score">0.9819795</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27353" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="612">
<infon key="score">0.9991903</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27381" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="613">
<infon key="score">0.9994134</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27431" length="2"/>
<text>CD</text>
</annotation>
<annotation id="614">
<infon key="score">0.88793606</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27444" length="6"/>
<text>unique</text>
</annotation>
<annotation id="615">
<infon key="score">0.9992924</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27451" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="616">
<infon key="score">0.99278283</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:44:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27529" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="617">
<infon key="score">0.9988718</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27595" length="11"/>
<text>active site</text>
</annotation>
<annotation id="618">
<infon key="score">0.99879134</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:40:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27630" length="20"/>
<text>Computer simulations</text>
</annotation>
<annotation id="619">
<infon key="score">0.95659685</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27691" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="620">
<infon key="score">0.999217</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27701" length="15"/>
<text>Ig-like domains</text>
</annotation>
<annotation id="621">
<infon key="score">0.99876976</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27813" length="11"/>
<text>active site</text>
</annotation>
<annotation id="622">
<infon key="score">0.99823534</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27849" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="623">
<infon key="score">0.5257192</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:40:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27871" length="8"/>
<text>modeling</text>
</annotation>
<annotation id="624">
<infon key="score">0.9989699</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:46:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27923" length="13"/>
<text>highly mobile</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>27958</offset>
<text>Methods</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>27966</offset>
<text>Cloning, Expression and Purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28003</offset>
<text>The gene encoding BlCel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Blcel5B_Fw and Blcel5B_Rv (Supplementary Table 3). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28384</offset>
<text>The same method was used for construction of domain deletions. For Ig-like + CBM46 deletion, Δ(Ig-CBM46), the fragment encoding the CD (nucleotides 82 to 1086) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1087-1683_Rv. For CBM46 deletion, ΔCBM46, the fragment encoding the CD + Ig-like (nucleotides 82 to 1377) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1378-1683_Rv (Supplementary Table 3). Both fragments were cloned into pETTRXA-1a/LIC.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28862</offset>
<text>The wt protein BlCel5B, mutated proteins and AM deletions were expressed in E. coli Rosetta2 (DE3) strain. The cells were grown at 37 °C and 150 RPM in Luria Bertani Broth medium supplemented with 50 μg/mL kanamycin to an A600 of 1.5–2.0, after which the temperature was reduced to 20 °C and protein expression was induced with 1 mM IPTG for 6 h.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>29224</offset>
<text>The extract was then loaded onto a NiNTA resin (Qiagen) equilibrated with a washing buffer (5 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Non-absorbed material was washed with ten times column volume with washing buffer and the purified protein was eluted with 200 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl at pH = 7.0. His6 tag was removed by overnight digestion with TEV (Tobacco Etch Virus) at 4 °C, and untagged protein was purified by gel filtration through a HiLoad 16/60 Superdex 200 column in buffer containing 50 mM NaCl, 25 mM Tris-HCl at pH 7.0.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>29812</offset>
<text>Site-directed Mutagenesis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>29838</offset>
<text>The BlCel5B point mutations W479A and W481A were obtained by the inverse PCR method of site-directed mutagenesis. Phusion® “High-Fidelity” DNA polymerase (NEB, USA) was used for amplifications with the plasmid pETTRXA-1a/LIC-Blcel5B as a template. Mutagenic primers Blcel5BW479A_Fw/Rv and Blcel5BW481A_Fw/Rv (Supplementary Table 3) were generated by HTP-OligoDesigner tool (http://www.ifsc.usp.br/htpoligo/).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>30252</offset>
<text>Activity Assays</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>30268</offset>
<text>Enzymatic activity assays were performed by a colorimetric method using the 3,5-dinitrosalicylic acid (DNS), with glucose being a standard for the calibration curves. Assays of optimal temperature and pH were performed in triplicate with 1% medium-viscosity CMC as the substrate. For optimal temperature, the reaction mixture containing 10 μL of enzyme at 0.1 mg/mL, 50 μL of 1% (w/v) CMC and 40 μL of 50 mM sodium citrate buffer (pH 5.0) was incubated at 30 to 80 °C for 15 min and stopped by adding 100 μL of DNS solution. After this, the mixture was incubated again for 5 min at 100 °C and the absorbance was measured at 540 nm with a spectrophotometer. For optimal pH determination, the same amount of enzyme and substrate were diluted in 40 mM acetate/borate/phosphate buffer (ABF) with different pH values ranging from 2.0 to 10.0. The reactions were carried out under the predetermined optimal temperature.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>31212</offset>
<text>The substrate specificity of the enzyme was determind using rye arabinan, xyloglucan, β-glucan, galactomannan, lichenan, β-mannan, Azo-Avicel and CMC as substrates. The substrates were diluted in water to 1% (w/v), and the reaction mixture was composed of 10 mL of purified enzyme at a concentration of 0.1 mg/mL, 0.4 mL of 50 mM sodium citrate buffer at pH 5.0, and 0.5 mL of 1% (w/v) substrate aqueous solution. The reaction was incubated at 50 °C for 15 min, followed by treatment with DNS as mentioned above. Enzyme unit was defined as the amount of enzyme that produces 1.0 μM of glucose in one minute for each substrate.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>31859</offset>
<text>The kinetic parameters were determined by increasing concentrations of CMC. Reactions were performed in 50 mM sodium citrate buffer (pH = 4.0) at 50 °C, and measured by DNS method as well. Kinetic constants were determined by non-linear regression using OriginPro 8.0.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32137</offset>
<text>Thermal Shift Assays</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32158</offset>
<text>The thermal denaturation assays were performed using a Real Time PCR Machine (Stratagene Mx3005P) as described by Dupeux and co-workers. Briefly, the enzymes were diluted to 10 μM in 50 mM sodium citrate buffer (pH = 4.0) containing 1x SYPRO orange dye (Thermo Fisher Scientific). The fluorescence emission of the probe was monitored (excitation and emission at 492 and 516 nm, respectively) varying the temperature between 25 and 75 °C with the rate of 1 °C/min.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32639</offset>
<text>Cellopentaose Cleavage Experiment</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32673</offset>
<text>The full-length BlCel5B and AM deletion constructs were tested for product formation from cellopentaose. Cellopentaose (1.0 mM) was incubated with 25 μg of purified enzyme in 10 mM ammonium bicarbonate buffer (pH 7.0) in a total volume of 50 μL. The reaction was incubated for 90 min at 50 °C and then stopped by treatment at 100 °C for 5 min. After centrifugation for 10 min at 16,000 g the samples were subjected to MALDI/TOF-MS. Samples were supplemented with NaCl to a final concentration of 20 mM and 1 μL of the supernatant was co-crystallized with 1 μL 2,5-dihydroxybenzoic acid (10 mg/mL) in acetonitrile 30% and spotted on the target plate. The products were analyzed on Microflex LT MALDI-TOF (Bruker Daltonics) operating in positive ion mode. A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.</text>
<annotation id="879">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:37:00Z</infon>
<location offset="33436" length="8"/>
<text>spectrum</text>
</annotation>
<annotation id="878">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:55Z</infon>
<location offset="33488" length="7"/>
<text>spectra</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>33575</offset>
<text>Crystallization, Data Collection, and Structure Determination</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>33637</offset>
<text>After purification, BlCel5B was concentrated to 10 mg/mL for crystallization trials. Crystallization screens were set up using the sitting-drop vapor-diffusion method on a Cartesian PixSys 4200 (Genomic Solutions, United Kingdom) in a 96-well plate with drops formed by 100 nL protein solution plus 100 nL reservoir solution. The commercial kits Crystal Screen and Index (Hampton) were used as initial conditions. Crystals were grown at 18 °C between 3 and 7 days, and screened for diffraction.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34141</offset>
<text>Crystals were supplemented with cryoprotection solution, flash cooled in liquid nitrogen and diffraction data were collected at 100 K, at beamline ID23-1 (wavelength of 0.97 Å) from the European Synchrotron Radiation Facility (Grenoble, France). A crystal grown in condition containing 22.5% PEG 4000, 14% isopropanol and 0.1 M sodium citrate, pH 6.0, was selected to collect diffraction data to 1.7 Å resolution. The complex of the enzyme with substrate was obtained by crystal soaking with five times molar excess of cellopentaose for 24 hours. Diffraction data for the complexed enzyme were collected at 1.75 Å resolution.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34782</offset>
<text>Data were integrated with iMosflm and scaled with Aimless. The structure was solved by molecular replacement with Phaser using an endoglucanase from Clostridium cellulovoran (PDB code: 3NDY) as the search model. Coot was used for density fitting, and refinement was performed with PHENIX.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>35071</offset>
<text>Atomistic simulations</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>35093</offset>
<text>We took the BlCel5B structure complexed with cellotetraose as the starting configuration for the MD simulations. The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server. Hydrogen atoms were then added according to the protonation states determined at the optimum pH of 4.0 using the H + + server. The following residues were considered protonated: H55, H77, D89, E96, E103, H114, E129, E159, E197, D198, E202, H205, E208, D211, H220, E245, E248, E260, H278, H292, D306, E312, E371, E375, E476, H416, E477, E489, D497, and E524. The remaining protonatable residues were considered in the standard protonation state. The BlCel5B-cellotetraose complex was then immersed in a rectangular simulation box of dimensions such that a solvent layer at least 16 Å thick surrounded the protein. The simulation box, built with Packmol, also contained 0.10 M NaCl aqueous solution with excess counter ions to keep the system electrically neutral. The final system comprised approximately 85500 atoms.</text>
<annotation id="891">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:43:11Z</infon>
<location offset="35247" length="3"/>
<text>apo</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>36152</offset>
<text>The simulations were performed using NAMD with the CHARMM force field and the TIP3P water model. Periodic boundary conditions were employed, using particle mesh Ewald to handle electrostatics and a 12-Å cutoff radius for truncating short-range potentials. Bonds involving hydrogen atoms were constrained at their equilibrium lengths and a time step of 2 fs was used to integrate the equations of motion. The simulations were carried out under constant pressure and temperature of 1 atm and 310 K, respectively, employing the Langevin barostat and thermostat.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>36714</offset>
<text>Accelerated Molecular Dynamics</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>36745</offset>
<text>In accelerated molecular dynamics, the trajectory is propagated on a modified potential aimed to enhance conformational sampling. Whenever the potential energy drops below a given threshold E, a boost ΔV(r) is applied, so that the escaping rates of local minima increase. When the potential energy gets over the threshold E, the system evolves on the original energy surface. This method has the advantage of conserving the general shape of the potential energy surface and of requiring no prior definition of reaction coordinates, so the system is allowed to explore freely its conformational space. Here, we restricted the energy boost only to the dihedral potential energy, as changes in torsion angles are the main source of conformational changes in proteins. The energy boost assumes the form of equation (1) that depends on the energy threshold E and on the parameter α – which modulates the shape of the potential energy surface where the boost is applied. We set the parameters E and α according to previous studies, which recommend that E equals the average dihedral energy obtained from a conventional MD simulation plus 4 kcal/mol times the number of residues, and α equals 0.8 kcal/mol times the number of residues. The average dihedral energy was 2275.5 kcal/mol and the BlCel5B has 516 residues, so we set E = 2275.5 + 4 × 516 = 4339.5 kcal/mol and α = 0.8 × 516 = 418.8 kcal/mol.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>38188</offset>
<text>Simulation procedures</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>38210</offset>
<text>BlCel5B-cellotetraose – Having built the system, we carried out the following steps for equilibration: (i) 1000 steps of energy minimization followed by 100 ps of MD simulation with all non-solvent heavy atoms fixed; (ii) same as (i), but with only the α carbons fixed; (iii) 5 ns of MD with all atoms free. After these preliminary steps, a trajectory lasting 400 ns was generated using conventional MD and then the aMD dihedral boost was applied for additional 1.0 μs. After 100 ns of conventional MD, the cellotetraose dissociated and the simulation began to represent the dynamics of unbound state of BlCel5B.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>38835</offset>
<text>BlCel5B-cellooctaose</text>
<annotation id="884">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:16Z</infon>
<location offset="38835" length="20"/>
<text>BlCel5B-cellooctaose</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>38856</offset>
<text>To build the cellooctaose chain, we extended the original cellotetraose chain in the crystal structure with 4 additional glucose residues spanning regions around the BlCel5B positive subsites. Then, we submitted the system to the following procedure: (i) 1000 steps of energy minimization followed by 1 ns of MD keeping all the non-solvent heavy atoms fixed, except the 4 modeled glucose residues of the cellooctaose chain; (ii) same as step (i), but with only the α carbons fixed; (iii) 1 ns of MD with only the non-modeled glucose residues fixed. We then performed a 200-ns-long MD with three harmonic potentials involving cellooctaose chain: first, between C3 atom (CHARMM atom names) of the second glucose residue from the cellooctaose non-reducing end and the CD2 atom of the W47 tryptophan residue; second, between the OH3 atom of the forth glucose residue from the cellooctaose non-reducing end and HE2 atom of the H113 histidine residue; and third, between the HE2 atom of the catalytic residue E159 and O4 glycosidic oxygen between the fourth and fifth glucose unit of the cellooctaose chain. After these preliminary relaxation steps, the harmonic potentials were removed and the trajectory was propagated by 400 ns using MD. To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain. After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation.</text>
<annotation id="885">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:16Z</infon>
<location offset="40113" length="20"/>
<text>BlCel5B-cellooctaose</text>
</annotation>
<annotation id="886">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:16Z</infon>
<location offset="40496" length="20"/>
<text>BlCel5B-cellooctaose</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>40605</offset>
<text>Coarse-grained MD simulations</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>40635</offset>
<text>The coarse-grained model was constructed from the minimized all-atom protein. We have used the domELNEDIN CG model for the protein. In this representation, an elastic network is used within each domain as a structural scaffold in order to maintain the overall shape of the protein, and a slightly modified version of MARTINI CG model describes the interactions involving beads not connected by harmonic springs.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>41047</offset>
<text>The delimitation of each domain was quite clear considering the short linkers connecting them and the recognition of their structural patterns in databases. We assumed CD, Ig-like module, and CBM46 as consisted of residues 18–331, 332–430, and 431–533, respectively. Therefore, there were elastic network bonds only within these domains (domELNEDIN CG model in Supplementary Fig. 6A).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>41438</offset>
<text>The protonation state of each residue bead in the protein was the same adopted in the atomistic simulations. The system was then solvated by 10000 standard MARTINI CG water beads, including 10% of antifreeze particles. Also, 58 chloride and 48 sodium ions were added for charge neutrality. The size of final system was 109 Å × 109 Å × 109 Å.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>41799</offset>
<text>Preliminary simulations were performed to test the elastic network (EN) parameters. We have tested six different ENs in 100 ns of simulations, using combinations of cut-off distance (Rc) of 8 Å and 9 Å with spring force constant (ks) of 500, 800 and 1000 kJ mol−1 nm−2. The time evolution of root mean square deviation relative to the crystal structure as well as the mobility profile of the protein in these simulations were compared to the correspondent data from a 100 ns atomistic simulation. From this procedure, the parameters Rc = 9 Å and ks = 500 kJ mol−1 nm−2 resulted in the best match between atomistic and coarse-grained simulations (Supplementary Fig. 6B).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>42500</offset>
<text>The coarse-grained simulations were carried out using GROMACS. Periodic boundary conditions were employed. Van der Waals interactions were shifted to zero in the range 0.9–1.2 nm, and the electrostatic interactions, in the range 0.0–1.2 nm. The simulations were performed in the isothermal-isobaric ensemble (NpT), employing the Berendsen thermostat and barostat for temperature and pressure control, respectively, with time constants τT = 0.5 ps and τp = 1.2 ps.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>42982</offset>
<text>The CG simulations were carried out using the following protocol: the system was first minimized for 1000 steps using the steepest descent method. Then, it was submitted to a relaxation procedure comprising gradual increasing in time step or temperature. In the first stage of relaxation, the protein beads were restrained with a 1000 kJ mol−1 nm−2 force constant and a 50 ps simulation was carried out at 50 K, using the short time step of 1 fs. In the second stage, the time step was increased up to 5 ps lasting 1000 ps of simulation time. In the last stage of relaxation, all the system is released to move and it underwent a gradual increase in temperature, consisting on five segments of 100 ps at 50, 100, 150, 200 and 310 K. After achieving the desired temperature of 310 K, we performed three production simulations using 20-fs timestep. We have used a random number generator for assigning velocities to generate three independent simulations.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>43950</offset>
<text>In general, smoothing of the energy surface in CG model makes the time scales faster. A speed up factor of 4 is typically employed to rescale the time scale of MARTINI CG systems. Therefore, all CG simulations times described here and in the main text are effective times, i.e., 4× simulation time.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>44250</offset>
<text>Small Angle X-ray Scattering</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>44279</offset>
<text>SAXS data were collected at the SAXS2 beamline of the Brazilian Synchrotron Light Laboratory-LNLS (Campinas, Brazil) on a bi-dimensional position sensitive CCD detector (MarResearch, USA) using the radiation wavelength 1.54 Å. The sample-detector distance of 1000 mm allowed covering the momentum transfer range 0.01 Å−1 < q < 0.35 Å−1 (q = 4πsin θ/λ, where 2θ is the scattering angle).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>44697</offset>
<text>The protein samples were prepared in McIlvaine’s buffer at 50 mM, pH 5 and 20 °C. In each measurement, two successive frames of 300s were recorded for each sample at 1 and 2 mg/mL to monitor radiation damage. The patterns were integrated using the FIT2D program. The comparative analysis for each scattering curve at 1 and 2 mg/mL of BlCel5B (data not shown), as well as the radius of gyration values (Rg), indicated that concentration and aggregation effects did not exist. The linearity of the Guinier plot indicated that the preparation was monodisperse.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>45266</offset>
<text>The radius of gyration of the molecules (Rg) was estimated by two methods, using the Guinier equation-I(q) = I(0).exp[(−q2.Rg2)/3], q.Rg < 1.3- and also with the inverse Fourier transform in GNOM. The same program was used to obtain the distance distribution function P(r) and the maximum diameter Dmax. Ten independent dummy atom models (DAMs) were restored by the ab initio proceeding implemented in DAMMIN package. The best model, selected using normalized spatial discrepancy parameter computed by DAMAVER program, was superimposed on the crystallographic model with the SUPCOMB.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>45860</offset>
<text>Then, based on the enzyme conformations reported by MD and protein crystallography, the computed X-ray scattering profile was fitted to a given experimental SAXS data by minimizing the χ function in the FOXS program.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>46079</offset>
<text>To assess the inter-domain information, the contribution of individual conformer and the flexibility of BlCel5B, we proceed in two approaches. First, the theoretical profiles and experimental data comparison was performed to infer the best-fit conformation of the ensemble-based analysis by the ensemble optimization method – EOM, which assumes coexistence of a range of conformations in solution for which an average scattering intensity fits the experimental SAXS data; all models were generated with the three individual domains (Ig-like, CBM46, and CD) free to randomly move in order to cover the entire conformational space. The second approach was based on a fractional volume calculation from three conformation members extracted from the MD simulations, each with a distinct scatter curve. OLIGOMER provided solution of a system of linear equations between the experimental and generated conformations by MD.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>46998</offset>
<text>The simulated scattering curves from the MD and crystallographic models were obtained using the CRYSOL.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>47102</offset>
<text>Phylogenetic assignment</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>47126</offset>
<text>Sequences for all GH5 members, in which only the catalytic domain were considered, were downloaded from PFAM database and their classification into subfamilies were obtained within the CAZy database. The sequences belonging to subfamily 4 were selected and those that had over 90% identity or represented partial coverage were rejected. Based on their multiple sequence alignment, the phylogenetic tree was constructed using the maximum likelihood method implemented in the MEGA program version 6.06. One hundred Bootstrap replications were performed to examine the reliability of the phylogenetic tree.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>47730</offset>
<text>Additional Information</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>47753</offset>
<text>How to cite this article: Liberato, M. V. et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. Rep. 6, 23473; doi: 10.1038/srep23473 (2016).</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>47959</offset>
<text>Supplementary Material</text>
</passage>
<passage>
<infon key="fpage">121</infon>
<infon key="lpage">45</infon>
<infon key="name_0">surname:Chundawat;given-names:S. P. S.</infon>
<infon key="name_1">surname:Beckham;given-names:G. T.</infon>
<infon key="name_2">surname:Himmel;given-names:M. E.</infon>
<infon key="name_3">surname:Dale;given-names:B. E.</infon>
<infon key="pub-id_pmid">22432613</infon>
<infon key="section_type">REF</infon>
<infon key="source">Annu. Rev. Chem. Biomol. Eng.</infon>
<infon key="type">ref</infon>
<infon key="volume">2</infon>
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<text>Author Contributions M.V.L. and I.P. designed the experiments; C.M.C. performed the gene mutations and cloning; M.V.L. and V.O.A.P. expressed and purified the enzymes and characterized the enzymatic activities; M.V.L. crystallized and determined the crystal structures, with data collection supervised by A.P.; M.A.K. performed mass spectrometry experiment; E.A.A. and M.O.N. collected and treated SAXS data. R.L.S., E.T.P. and M.S.S. designed the computer simulations; R.L.S. performed Accelerated Molecular Dynamics; E.T.P. performed Coarse-grained MD Simulations; M.V.L., R.L.S., M.S.S. and I.P. wrote the manuscript with the input from all the other authors; M.S.S. and I.P. supervised the project.</text>
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<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>53941</offset>
<text>Crystal models of BlCel5B.</text>
<annotation id="625">
<infon key="score">0.99844503</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:35:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53941" length="14"/>
<text>Crystal models</text>
</annotation>
<annotation id="626">
<infon key="score">0.99846846</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53959" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>53968</offset>
<text>Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b). The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue). Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow). The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.</text>
<annotation id="627">
<infon key="score">0.99364626</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53977" length="9"/>
<text>structure</text>
</annotation>
<annotation id="797">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<location offset="54069" length="2"/>
<text>CD</text>
</annotation>
<annotation id="628">
<infon key="score">0.99158597</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:35:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54099" length="15"/>
<text>TIM-barrel fold</text>
</annotation>
<annotation id="629">
<infon key="score">0.9988049</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:45:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54124" length="22"/>
<text>substrate-binding site</text>
</annotation>
<annotation id="630">
<infon key="score">0.76680785</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54162" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="631">
<infon key="score">0.99875355</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54205" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="632">
<infon key="score">0.88893646</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54253" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="633">
<infon key="score">0.9994466</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54268" length="4"/>
<text>W479</text>
</annotation>
<annotation id="634">
<infon key="score">0.99945384</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54277" length="4"/>
<text>W481</text>
</annotation>
<annotation id="635">
<infon key="score">0.9993051</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54313" length="13"/>
<text>cellotetraose</text>
</annotation>
<annotation id="636">
<infon key="score">0.99909556</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54341" length="14"/>
<text>Ig-like domain</text>
</annotation>
<annotation id="637">
<infon key="score">0.9991934</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54423" length="2"/>
<text>CD</text>
</annotation>
<annotation id="638">
<infon key="score">0.8081565</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54430" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="639">
<infon key="score">0.99850583</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54443" length="13"/>
<text>superposition</text>
</annotation>
<annotation id="640">
<infon key="score">0.99915457</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:49:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54464" length="14"/>
<text>Ig-like domain</text>
</annotation>
<annotation id="641">
<infon key="score">0.9668259</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54483" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="642">
<infon key="score">0.999071</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54585" length="4"/>
<text>loop</text>
</annotation>
<annotation id="643">
<infon key="score">0.999288</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54623" length="13"/>
<text>Cellotetraose</text>
</annotation>
<annotation id="644">
<infon key="score">0.93913716</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="54646" length="17"/>
<text>subsites -1 to -3</text>
</annotation>
<annotation id="832">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:57Z</infon>
<location offset="54681" length="11"/>
<text>coordinated</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>54730</offset>
<text>BlCel5B enzymatic activity characterization.</text>
<annotation id="645">
<infon key="score">0.99579805</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54730" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="877">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:47Z</infon>
<location offset="54738" length="35"/>
<text>enzymatic activity characterization</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>54775</offset>
<text>(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5). The first three spectra show the substrate, enzyme and buffer controls. The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2. The last two spectra show that the C-terminal deletions eliminate the enzyme activity. BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively. The enzyme exhibits optimal pH of 4.0 and optimal temperature of 55 °C, retaining about 50% of its activity at 80 °C. (d) Michaelis-Menten curve using CMC as a substrate.</text>
<annotation id="646">
<infon key="score">0.9980982</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54779" length="12"/>
<text>MALDI/TOF-MS</text>
</annotation>
<annotation id="647">
<infon key="score">0.9872946</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54792" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="648">
<infon key="score">0.99913824</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54845" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="883">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:38:40Z</infon>
<location offset="54865" length="19"/>
<text>deletion constructs</text>
</annotation>
<annotation id="649">
<infon key="score">0.9989943</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54886" length="6"/>
<text>ΔCBM46</text>
</annotation>
<annotation id="650">
<infon key="score">0.99894</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:06:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54897" length="9"/>
<text>ΔIg-CBM46</text>
</annotation>
<annotation id="651">
<infon key="score">0.99934024</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54927" length="13"/>
<text>cellopentaose</text>
</annotation>
<annotation id="652">
<infon key="score">0.97373194</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54942" length="2"/>
<text>C5</text>
</annotation>
<annotation id="653">
<infon key="score">0.86749804</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54963" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="654">
<infon key="score">0.7187502</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55029" length="8"/>
<text>spectrum</text>
</annotation>
<annotation id="655">
<infon key="score">0.9990318</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:37:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55051" length="11"/>
<text>full length</text>
</annotation>
<annotation id="656">
<infon key="score">0.9991542</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55063" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="657">
<infon key="score">0.86857474</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55109" length="2"/>
<text>C5</text>
</annotation>
<annotation id="658">
<infon key="score">0.9976169</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:53:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55125" length="16"/>
<text>oligosaccharides</text>
</annotation>
<annotation id="659">
<infon key="score">0.50656426</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55150" length="2"/>
<text>C4</text>
</annotation>
<annotation id="880">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:37:19Z</infon>
<location offset="55154" length="2"/>
<text>C3</text>
</annotation>
<annotation id="881">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:37:32Z</infon>
<location offset="55161" length="2"/>
<text>C2</text>
</annotation>
<annotation id="660">
<infon key="score">0.83065146</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:36:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55178" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="882">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:38:25Z</infon>
<location offset="55221" length="29"/>
<text>eliminate the enzyme activity</text>
</annotation>
<annotation id="661">
<infon key="score">0.9991924</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55252" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="662">
<infon key="score">0.99763143</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55274" length="3"/>
<text>CMC</text>
</annotation>
<annotation id="663">
<infon key="score">0.99861073</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:37:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55475" length="22"/>
<text>Michaelis-Menten curve</text>
</annotation>
<annotation id="664">
<infon key="score">0.9977623</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:03:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55504" length="3"/>
<text>CMC</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>55532</offset>
<text>Open-close transitions of BlCel5B.</text>
<annotation id="665">
<infon key="score">0.752593</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55532" length="4"/>
<text>Open</text>
</annotation>
<annotation id="666">
<infon key="score">0.92618257</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:21:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55537" length="5"/>
<text>close</text>
</annotation>
<annotation id="667">
<infon key="score">0.9987601</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55558" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>55567</offset>
<text>(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations. The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose). For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise). (d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4. Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.</text>
<annotation id="668">
<infon key="score">0.9991435</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55571" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="824">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:59:48Z</infon>
<location offset="55586" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="669">
<infon key="score">0.9991783</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55622" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="670">
<infon key="score">0.99471444</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55634" length="12"/>
<text>cellooctaose</text>
</annotation>
<annotation id="849">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:12:40Z</infon>
<location offset="55667" length="15"/>
<text>aMD simulations</text>
</annotation>
<annotation id="846">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<location offset="55688" length="8"/>
<text>distance</text>
</annotation>
<annotation id="671">
<infon key="score">0.9995372</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55730" length="4"/>
<text>I120</text>
</annotation>
<annotation id="672">
<infon key="score">0.9992906</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="55736" length="2"/>
<text>CD</text>
</annotation>
<annotation id="673">
<infon key="score">0.99951124</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55744" length="4"/>
<text>E477</text>
</annotation>
<annotation id="674">
<infon key="score">0.99878496</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="55750" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="847">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<location offset="55854" length="8"/>
<text>distance</text>
</annotation>
<annotation id="675">
<infon key="score">0.99880093</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55881" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="676">
<infon key="score">0.9990532</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55909" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="677">
<infon key="score">0.9680609</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:11:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55921" length="12"/>
<text>cellooctaose</text>
</annotation>
<annotation id="678">
<infon key="score">0.99882704</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55944" length="14"/>
<text>substrate-free</text>
</annotation>
<annotation id="679">
<infon key="score">0.99827063</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56002" length="3"/>
<text>aMD</text>
</annotation>
<annotation id="680">
<infon key="score">0.9992008</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:15Z</infon>
<infon key="identifier">GO:</infon>
<location offset="56015" length="20"/>
<text>BlCel5B-cellooctaose</text>
</annotation>
<annotation id="681">
<infon key="score">0.9983491</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56072" length="3"/>
<text>aMD</text>
</annotation>
<annotation id="682">
<infon key="score">0.99800247</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56124" length="2"/>
<text>MD</text>
</annotation>
<annotation id="683">
<infon key="score">0.99917036</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="56165" length="20"/>
<text>BlCel5B-cellooctaose</text>
</annotation>
<annotation id="684">
<infon key="score">0.9969229</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56212" length="10"/>
<text>tryptophan</text>
</annotation>
<annotation id="685">
<infon key="score">0.9684056</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:53:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="56255" length="6"/>
<text>glucan</text>
</annotation>
<annotation id="686">
<infon key="score">0.90202075</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56271" length="17"/>
<text>subsites −4 to +4</text>
</annotation>
<annotation id="687">
<infon key="score">0.99953365</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56299" length="4"/>
<text>W479</text>
</annotation>
<annotation id="688">
<infon key="score">0.9995183</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56308" length="4"/>
<text>W481</text>
</annotation>
<annotation id="689">
<infon key="score">0.9978777</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56323" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="690">
<infon key="score">0.9992914</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56389" length="6"/>
<text>closed</text>
</annotation>
<annotation id="691">
<infon key="score">0.9992299</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="56413" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f4.jpg</infon>
<infon key="id">f4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>56441</offset>
<text>Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.</text>
<annotation id="692">
<infon key="score">0.99787855</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="56466" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="693">
<infon key="score">0.998811</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56486" length="13"/>
<text>superposition</text>
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<annotation id="694">
<infon key="score">0.9975733</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56547" length="4"/>
<text>SAXS</text>
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<annotation id="695">
<infon key="score">0.9919932</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56552" length="8"/>
<text>envelope</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f4.jpg</infon>
<infon key="id">f4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>56562</offset>
<text>(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations. Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III). (d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.</text>
<annotation id="696">
<infon key="score">0.9991328</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="56566" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="697">
<infon key="score">0.9950984</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:39:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56574" length="9"/>
<text>structure</text>
</annotation>
<annotation id="698">
<infon key="score">0.70716023</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56596" length="8"/>
<text>distance</text>
</annotation>
<annotation id="699">
<infon key="score">0.9995233</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56644" length="4"/>
<text>I120</text>
</annotation>
<annotation id="700">
<infon key="score">0.99948287</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56653" length="4"/>
<text>E477</text>
</annotation>
<annotation id="701">
<infon key="score">0.9993231</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56690" length="2"/>
<text>CD</text>
</annotation>
<annotation id="702">
<infon key="score">0.9975872</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56697" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="703">
<infon key="score">0.97517735</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56808" length="4"/>
<text>I120</text>
</annotation>
<annotation id="704">
<infon key="score">0.9865943</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:14:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56813" length="4"/>
<text>E477</text>
</annotation>
<annotation id="705">
<infon key="score">0.958231</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56818" length="8"/>
<text>distance</text>
</annotation>
<annotation id="887">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:40:10Z</infon>
<location offset="56842" length="17"/>
<text>CG-MD simulations</text>
</annotation>
<annotation id="706">
<infon key="score">0.9907962</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56932" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="707">
<infon key="score">0.9977046</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:40:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56977" length="21"/>
<text>distance distribution</text>
</annotation>
<annotation id="708">
<infon key="score">0.9993717</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57028" length="6"/>
<text>closed</text>
</annotation>
<annotation id="709">
<infon key="score">0.99937314</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57038" length="8"/>
<text>occluded</text>
</annotation>
<annotation id="710">
<infon key="score">0.9986526</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="57047" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="711">
<infon key="score">0.9991204</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="57053" length="2"/>
<text>CD</text>
</annotation>
<annotation id="712">
<infon key="score">0.9991974</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57075" length="9"/>
<text>semi-open</text>
</annotation>
<annotation id="853">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:43Z</infon>
<location offset="57115" length="26"/>
<text>crystallographic structure</text>
</annotation>
<annotation id="713">
<infon key="score">0.9992988</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57147" length="8"/>
<text>extended</text>
</annotation>
<annotation id="714">
<infon key="score">0.99879897</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57178" length="15"/>
<text>Superimposition</text>
</annotation>
<annotation id="715">
<infon key="score">0.9991222</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="57249" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="716">
<infon key="score">0.99679524</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57266" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="717">
<infon key="score">0.43775445</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57271" length="5"/>
<text>model</text>
</annotation>
<annotation id="718">
<infon key="score">0.5059148</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57290" length="10"/>
<text>structures</text>
</annotation>
<annotation id="719">
<infon key="score">0.94961184</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:40:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57319" length="10"/>
<text>simulation</text>
</annotation>
<annotation id="720">
<infon key="score">0.9985703</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:40:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57404" length="10"/>
<text>superposed</text>
</annotation>
<annotation id="721">
<infon key="score">0.99070346</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57422" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="722">
<infon key="score">0.8086411</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:16:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57427" length="8"/>
<text>envelope</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f5.jpg</infon>
<infon key="id">f5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>57440</offset>
<text>Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.</text>
<annotation id="888">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:05Z</infon>
<location offset="57440" length="10"/>
<text>Comparison</text>
</annotation>
<annotation id="723">
<infon key="score">0.99894464</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="57458" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="819">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:47:14Z</infon>
<location offset="57480" length="5"/>
<text>GH5_4</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f5.jpg</infon>
<infon key="id">f5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>57530</offset>
<text>(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).</text>
<annotation id="724">
<infon key="score">0.9975522</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="57534" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="889">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:13Z</infon>
<location offset="57549" length="16"/>
<text>crystallographic</text>
</annotation>
<annotation id="725">
<infon key="score">0.99914753</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="57570" length="6"/>
<text>closed</text>
</annotation>
<annotation id="726">
<infon key="score">0.9984163</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:47:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57596" length="19"/>
<text>Bacillus halodurans</text>
</annotation>
<annotation id="727">
<infon key="score">0.99806625</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:48:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="57616" length="5"/>
<text>Cel5B</text>
</annotation>
<annotation id="728">
<infon key="score">0.9970697</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="57623" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="729">
<infon key="score">0.9984063</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57651" length="21"/>
<text>Piromyces rhizinflata</text>
</annotation>
<annotation id="730">
<infon key="score">0.9514452</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57673" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="731">
<infon key="score">0.9991021</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57677" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="732">
<infon key="score">0.9984065</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57711" length="26"/>
<text>Clostridium cellulolyticum</text>
</annotation>
<annotation id="733">
<infon key="score">0.9659778</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
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<text>GH5</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57742" length="13"/>
<text>endoglucanase</text>
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<infon key="score">0.9983325</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57776" length="25"/>
<text>Clostridium cellulovorans</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57802" length="3"/>
<text>GH5</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57806" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="738">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57840" length="18"/>
<text>Bacteroides ovatus</text>
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<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57859" length="3"/>
<text>GH5</text>
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<annotation id="740">
<infon key="score">0.9991406</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57863" length="13"/>
<text>xyloglucanase</text>
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<annotation id="741">
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<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57897" length="20"/>
<text>Paenibacillus pabuli</text>
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<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57918" length="3"/>
<text>GH5</text>
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<infon key="score">0.9991715</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57922" length="13"/>
<text>xyloglucanase</text>
</annotation>
<annotation id="744">
<infon key="score">0.9983506</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57956" length="19"/>
<text>Prevotella bryantii</text>
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<annotation id="745">
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<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57976" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="746">
<infon key="score">0.9989398</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:48:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="57980" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="747">
<infon key="score">0.99841285</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58014" length="30"/>
<text>Ruminiclostridium thermocellum</text>
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<annotation id="748">
<infon key="score">0.8743299</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:46:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58061" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="749">
<infon key="score">0.9991216</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58065" length="9"/>
<text>cellulase</text>
</annotation>
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<infon key="score">0.99889106</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58076" length="8"/>
<text>xylanase</text>
</annotation>
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<infon key="score">0.9972579</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58089" length="7"/>
<text>mannase</text>
</annotation>
<annotation id="752">
<infon key="score">0.7350698</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:41:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58117" length="23"/>
<text>Bacteroidetes bacterium</text>
</annotation>
<annotation id="753">
<infon key="score">0.7986666</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58141" length="4"/>
<text>AC2a</text>
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<annotation id="754">
<infon key="score">0.9983577</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58146" length="13"/>
<text>endocellulase</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f6.jpg</infon>
<infon key="id">f6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>58176</offset>
<text>Comparison of the binding cleft of the BlCel5B and BhCel5B.</text>
<annotation id="890">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:33Z</infon>
<location offset="58176" length="10"/>
<text>Comparison</text>
</annotation>
<annotation id="755">
<infon key="score">0.9991272</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58194" length="13"/>
<text>binding cleft</text>
</annotation>
<annotation id="756">
<infon key="score">0.9568515</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58215" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="757">
<infon key="score">0.9822322</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58227" length="7"/>
<text>BhCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f6.jpg</infon>
<infon key="id">f6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>58236</offset>
<text>The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185. We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.</text>
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<infon key="score">0.9989668</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58264" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="759">
<infon key="score">0.99901545</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58276" length="7"/>
<text>BhCel5B</text>
</annotation>
<annotation id="760">
<infon key="score">0.9974975</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:42:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58321" length="5"/>
<text>cleft</text>
</annotation>
<annotation id="829">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:01:00Z</infon>
<location offset="58338" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="761">
<infon key="score">0.9995627</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58358" length="4"/>
<text>W181</text>
</annotation>
<annotation id="762">
<infon key="score">0.9989575</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:53:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58370" length="4"/>
<text>loop</text>
</annotation>
<annotation id="763">
<infon key="score">0.99955577</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58383" length="4"/>
<text>F177</text>
</annotation>
<annotation id="764">
<infon key="score">0.9995542</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:36:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58392" length="4"/>
<text>R185</text>
</annotation>
<annotation id="765">
<infon key="score">0.99892116</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:35:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58440" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="766">
<infon key="score">0.9979352</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58501" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="767">
<infon key="score">0.9990663</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58520" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f7.jpg</infon>
<infon key="id">f7</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>58549</offset>
<text>Proposed molecular mechanism of BlCel5B conformational selection.</text>
<annotation id="768">
<infon key="score">0.9990453</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58581" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">srep23473-f7.jpg</infon>
<infon key="id">f7</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>58615</offset>
<text>As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states. In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments. After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.</text>
<annotation id="769">
<infon key="score">0.9985917</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58635" length="11"/>
<text>simulations</text>
</annotation>
<annotation id="770">
<infon key="score">0.99830294</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:52:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="58651" length="4"/>
<text>SAXS</text>
</annotation>
<annotation id="771">
<infon key="score">0.99919575</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58662" length="7"/>
<text>BlCel5B</text>
</annotation>
<annotation id="772">
<infon key="score">0.9993125</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:10:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58712" length="6"/>
<text>closed</text>
</annotation>
<annotation id="773">
<infon key="score">0.9993204</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:17:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58722" length="8"/>
<text>extended</text>
</annotation>
<annotation id="774">
<infon key="score">0.9992793</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58731" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="775">
<infon key="score">0.99938405</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58737" length="2"/>
<text>CD</text>
</annotation>
<annotation id="776">
<infon key="score">0.9992828</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T10:13:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58759" length="4"/>
<text>open</text>
</annotation>
<annotation id="777">
<infon key="score">0.99899673</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58799" length="11"/>
<text>active site</text>
</annotation>
<annotation id="778">
<infon key="score">0.9981187</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58850" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="779">
<infon key="score">0.99932384</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="58861" length="2"/>
<text>CD</text>
</annotation>
<annotation id="780">
<infon key="score">0.99932706</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:43:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="58968" length="3"/>
<text>apo</text>
</annotation>
<annotation id="781">
<infon key="score">0.99923134</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="58972" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_title_caption</infon>
<offset>59018</offset>
<text>Activity of BlCel5B constructs against tested substrates.</text>
<annotation id="782">
<infon key="score">0.9987154</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T09:40:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="59030" length="7"/>
<text>BlCel5B</text>
</annotation>
</passage>
<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
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<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="bottom" charoff="50">Substrate (1%)</th><th colspan="5" align="center" valign="top" charoff="50">Relative Activity (%)</th></tr><tr><th align="center" valign="top" charoff="50">WT<xref ref-type="fn" rid="t1-fn1">*</xref></th><th align="center" valign="top" charoff="50">W479A</th><th align="center" valign="top" charoff="50">W481A</th><th align="center" valign="top" charoff="50">ΔCBM46</th><th align="center" valign="top" charoff="50">ΔIg-CBM46</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">β-glucan</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">79.1</td><td align="center" valign="top" charoff="50">63.6</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">CMC</td><td align="center" valign="top" charoff="50">25.5</td><td align="center" valign="top" charoff="50">12.2</td><td align="center" valign="top" charoff="50">2.4</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Lichenan</td><td align="center" valign="top" charoff="50">52.4</td><td align="center" valign="top" charoff="50">41</td><td align="center" valign="top" charoff="50">28.6</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Xyloglucan</td><td align="center" valign="top" charoff="50">45.2</td><td align="center" valign="top" charoff="50">41.2</td><td align="center" valign="top" charoff="50">30.8</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Azo-Avicel</td><td align="center" valign="top" charoff="50">nd<xref ref-type="fn" rid="t1-fn2">**</xref></td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Arabinoxylan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Galactomannan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">1,4-β-mannan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr></tbody></table>
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<text>Substrate (1%) Relative Activity (%) WT* W479A W481A ΔCBM46 ΔIg-CBM46 β-glucan 100 79.1 63.6 nd nd CMC 25.5 12.2 2.4 nd nd Lichenan 52.4 41 28.6 nd nd Xyloglucan 45.2 41.2 30.8 nd nd Azo-Avicel nd** nd nd nd nd Arabinoxylan nd nd nd nd nd Galactomannan nd nd nd nd nd 1,4-β-mannan nd nd nd nd nd </text>
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<text>W481A</text>
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<text>ΔCBM46</text>
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<infon key="type">mutant</infon>
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<text>ΔIg-CBM46</text>
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<text>β-glucan</text>
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<infon key="type">chemical</infon>
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<text>CMC</text>
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<text>Lichenan</text>
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<text>Xyloglucan</text>
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<text>Azo-Avicel</text>
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<text>Arabinoxylan</text>
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<infon key="score">0.99856454</infon>
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<text>Galactomannan</text>
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<infon key="score">0.99896324</infon>
<infon key="type">chemical</infon>
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<infon key="updated_at">2023-09-21T10:04:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="59362" length="12"/>
<text>1,4-β-mannan</text>
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<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>59407</offset>
<text>*WT = wild type.</text>
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<infon key="score">0.90642285</infon>
<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-21T10:08:45Z</infon>
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<location offset="59408" length="2"/>
<text>WT</text>
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<annotation id="792">
<infon key="score">0.99874187</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-21T13:45:23Z</infon>
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<text>wild type</text>
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<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>59428</offset>
<text>**nd = not detected.</text>
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</document>
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|
<?xml version="1.0" encoding="UTF-8"?>
<!DOCTYPE collection SYSTEM "BioC.dtd">
<collection>
<source>PMC</source>
<date>20230107</date>
<key>pmc.key</key>
<document>
<id>4980666</id>
<infon key="license">CC BY</infon>
<infon key="tt_curatable">no</infon>
<infon key="tt_version">2</infon>
<infon key="tt_round">2</infon>
<passage>
<infon key="article-id_doi">10.1038/srep31500</infon>
<infon key="article-id_pii">srep31500</infon>
<infon key="article-id_pmc">4980666</infon>
<infon key="article-id_pmid">27510745</infon>
<infon key="elocation-id">31500</infon>
<infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
<infon key="name_0">surname:Carcelli;given-names:Mauro</infon>
<infon key="name_1">surname:Rogolino;given-names:Dominga</infon>
<infon key="name_2">surname:Gatti;given-names:Anna</infon>
<infon key="name_3">surname:De Luca;given-names:Laura</infon>
<infon key="name_4">surname:Sechi;given-names:Mario</infon>
<infon key="name_5">surname:Kumar;given-names:Gyanendra</infon>
<infon key="name_6">surname:White;given-names:Stephen W.</infon>
<infon key="name_7">surname:Stevaert;given-names:Annelies</infon>
<infon key="name_8">surname:Naesens;given-names:Lieve</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">6</infon>
<infon key="year">2016</infon>
<offset>0</offset>
<text>N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes</text>
<annotation id="1">
<infon key="score">0.9991147</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="0" length="15"/>
<text>N-acylhydrazone</text>
</annotation>
<annotation id="605">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:45Z</infon>
<location offset="30" length="9"/>
<text>influenza</text>
</annotation>
<annotation id="585">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="40" length="5"/>
<text>virus</text>
</annotation>
<annotation id="524">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:00Z</infon>
<location offset="46" length="2"/>
<text>PA</text>
</annotation>
<annotation id="516">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<location offset="49" length="12"/>
<text>endonuclease</text>
</annotation>
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<passage>
<infon key="section_type">ABSTRACT</infon>
<infon key="type">abstract</infon>
<offset>97</offset>
<text>Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.</text>
<annotation id="606">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:45Z</infon>
<location offset="97" length="9"/>
<text>Influenza</text>
</annotation>
<annotation id="586">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="107" length="5"/>
<text>virus</text>
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<annotation id="525">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:00Z</infon>
<location offset="113" length="2"/>
<text>PA</text>
</annotation>
<annotation id="517">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<location offset="116" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="2">
<infon key="score">0.9991626</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="273" length="4"/>
<text>Mg2+</text>
</annotation>
<annotation id="570">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:17Z</infon>
<location offset="281" length="4"/>
<text>Mn2+</text>
</annotation>
<annotation id="3">
<infon key="score">0.9989165</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="294" length="14"/>
<text>catalytic site</text>
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<annotation id="644">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:48Z</infon>
<location offset="310" length="9"/>
<text>chelation</text>
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<infon key="score">0.99873513</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="445" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
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<infon key="score">0.9988744</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="468" length="15"/>
<text>enzymatic assay</text>
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<annotation id="526">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="489" length="2"/>
<text>PA</text>
</annotation>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="492" length="4"/>
<text>Nter</text>
</annotation>
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<infon key="score">0.9952571</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="497" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="581">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:35Z</infon>
<location offset="525" length="40"/>
<text>cell-based influenza vRNP reconstitution</text>
</annotation>
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<infon key="score">0.8346987</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="570" length="18"/>
<text>virus yield assays</text>
</annotation>
<annotation id="8">
<infon key="score">0.998764</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="598" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="571">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<location offset="649" length="9"/>
<text>influenza</text>
</annotation>
<annotation id="9">
<infon key="score">0.9989174</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:55:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="732" length="29"/>
<text>Computational docking studies</text>
</annotation>
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<infon key="score">0.9984523</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="842" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="11">
<infon key="score">0.9986233</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="865" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="12">
<infon key="score">0.9964547</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="914" length="17"/>
<text>crystal structure</text>
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<annotation id="527">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="935" length="2"/>
<text>PA</text>
</annotation>
<annotation id="549">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="938" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="13">
<infon key="score">0.9980684</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="943" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="14">
<infon key="score">0.9990225</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1042" length="11"/>
<text>active site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1057</offset>
<text>Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae. Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009. Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir. Therefore, there is an urgent need for new antiviral drugs with an entirely different mode of action.</text>
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<text>Orthomyxoviridae</text>
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<text>B</text>
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<text>influenza</text>
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<text>influenza</text>
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<text>viral</text>
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<text>amantadine</text>
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<infon key="updated_at">2023-09-20T15:47:22Z</infon>
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<text>viral</text>
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<infon key="updated_at">2023-09-20T15:48:13Z</infon>
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<text>M2</text>
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<text>virus</text>
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<text>oseltamivir</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1984</offset>
<text>The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA. The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.</text>
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<text>virus</text>
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<location offset="2004" length="10"/>
<text>polymerase</text>
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<text>PB2</text>
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<infon key="updated_at">2023-09-20T15:42:01Z</infon>
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<text>PA</text>
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<text>subunit</text>
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<text>endonuclease</text>
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<text>subunit</text>
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<location offset="2196" length="11"/>
<text>capped RNAs</text>
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<text>RNA</text>
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<infon key="updated_at">2023-09-20T15:49:39Z</infon>
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<text>PB1</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2277</offset>
<text>Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.</text>
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<text>viral</text>
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<text>influenza</text>
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<text>virus</text>
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<text>polymerase</text>
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<text>PA</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2528</offset>
<text>The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195). It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions. The two-metal-ion model is consistent with numerous biochemical findings. Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant. A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase. HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).</text>
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<location offset="2532" length="12"/>
<text>endonuclease</text>
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<infon key="updated_at">2023-09-20T15:44:10Z</infon>
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<text>catalytic site</text>
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<text>N-terminal domain</text>
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<text>PA</text>
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<text>PA</text>
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<text>Nter</text>
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<infon key="updated_at">2023-09-20T15:51:28Z</infon>
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<text>1~195</text>
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<infon key="updated_at">2023-09-20T15:51:33Z</infon>
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<text>histidine</text>
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<infon key="updated_at">2023-09-20T15:51:36Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>His41</text>
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<infon key="updated_at">2023-09-20T15:51:41Z</infon>
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<location offset="2682" length="18"/>
<text>strictly conserved</text>
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<infon key="updated_at">2023-09-20T15:51:43Z</infon>
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<location offset="2701" length="6"/>
<text>acidic</text>
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<infon key="updated_at">2023-09-20T15:51:47Z</infon>
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<location offset="2718" length="5"/>
<text>Glu80</text>
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<location offset="2725" length="6"/>
<text>Asp108</text>
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<infon key="identifier">DUMMY:</infon>
<location offset="2733" length="6"/>
<text>Glu119</text>
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<infon key="updated_at">2023-09-20T15:51:58Z</infon>
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<text>coordinate</text>
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<text>In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication. N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus. In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3). Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity. Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.</text>
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<infon key="updated_at">2023-09-20T15:54:58Z</infon>
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<text>EC50</text>
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<infon key="updated_at">2023-09-20T15:55:35Z</infon>
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<text>active site</text>
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<infon key="updated_at">2023-09-20T15:56:46Z</infon>
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<infon key="updated_at">2023-09-20T15:45:13Z</infon>
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<text>in complex with</text>
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<passage>
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<offset>4797</offset>
<text>Results and Discussion</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>4820</offset>
<text>Chemistry</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>4830</offset>
<text>N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis. Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum. Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).</text>
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<text>N-acylhydrazones</text>
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<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-20T15:57:29Z</infon>
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<text>mass spectrometry</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5003" length="18"/>
<text>elemental analysis</text>
</annotation>
<annotation id="96">
<infon key="score">0.90843266</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5076" length="4"/>
<text>1–27</text>
</annotation>
<annotation id="97">
<infon key="score">0.9943388</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5198" length="6"/>
<text>1H-NMR</text>
</annotation>
<annotation id="621">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<location offset="5205" length="8"/>
<text>spectrum</text>
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<annotation id="98">
<infon key="score">0.8405048</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5267" length="1"/>
<text>3</text>
</annotation>
<annotation id="99">
<infon key="score">0.9325075</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5273" length="1"/>
<text>4</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>5326</offset>
<text>If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4). Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A). In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4). In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized. Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety. Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.</text>
<annotation id="100">
<infon key="score">0.99670947</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5400" length="14"/>
<text>acylhydrazones</text>
</annotation>
<annotation id="101">
<infon key="score">0.87742805</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5419" length="10"/>
<text>coordinate</text>
</annotation>
<annotation id="102">
<infon key="score">0.9988938</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:16Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5521" length="43"/>
<text>N’-(2,3-dihydroxybenzylidene)-semicarbazide</text>
</annotation>
<annotation id="103">
<infon key="score">0.9352348</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5566" length="1"/>
<text>1</text>
</annotation>
<annotation id="104">
<infon key="score">0.92682606</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5595" length="1"/>
<text>2</text>
</annotation>
<annotation id="105">
<infon key="score">0.92472315</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5646" length="3"/>
<text>3–8</text>
</annotation>
<annotation id="106">
<infon key="score">0.9970642</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5651" length="2"/>
<text>18</text>
</annotation>
<annotation id="107">
<infon key="score">0.9953459</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5655" length="2"/>
<text>19</text>
</annotation>
<annotation id="108">
<infon key="score">0.9968497</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5672" length="2"/>
<text>18</text>
</annotation>
<annotation id="109">
<infon key="score">0.9944232</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5679" length="2"/>
<text>19</text>
</annotation>
<annotation id="110">
<infon key="score">0.9775343</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5692" length="6"/>
<text>gallic</text>
</annotation>
<annotation id="645">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:48Z</infon>
<location offset="5729" length="9"/>
<text>chelation</text>
</annotation>
<annotation id="111">
<infon key="score">0.84397453</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5838" length="4"/>
<text>9–11</text>
</annotation>
<annotation id="112">
<infon key="score">0.8735382</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5844" length="5"/>
<text>13–17</text>
</annotation>
<annotation id="113">
<infon key="score">0.96470314</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5851" length="5"/>
<text>20–23</text>
</annotation>
<annotation id="114">
<infon key="score">0.99696344</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5861" length="2"/>
<text>27</text>
</annotation>
<annotation id="115">
<infon key="score">0.9877781</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5896" length="2"/>
<text>12</text>
</annotation>
<annotation id="116">
<infon key="score">0.9775613</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5964" length="6"/>
<text>gallic</text>
</annotation>
<annotation id="117">
<infon key="score">0.9980586</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:58:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5988" length="2"/>
<text>26</text>
</annotation>
<annotation id="118">
<infon key="score">0.8888434</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:45:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6042" length="3"/>
<text>HIV</text>
</annotation>
<annotation id="119">
<infon key="score">0.99612683</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6046" length="7"/>
<text>RNase H</text>
</annotation>
<annotation id="120">
<infon key="score">0.9988881</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:52:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6079" length="9"/>
<text>magnesium</text>
</annotation>
<annotation id="121">
<infon key="score">0.9984977</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6101" length="11"/>
<text>active site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>6128</offset>
<text>Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+. Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized. The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex. In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion. This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion. ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.</text>
<annotation id="122">
<infon key="score">0.9984599</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6165" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="646">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:48Z</infon>
<location offset="6202" length="9"/>
<text>chelation</text>
</annotation>
<annotation id="123">
<infon key="score">0.93400145</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6228" length="5"/>
<text>metal</text>
</annotation>
<annotation id="124">
<infon key="score">0.9970854</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6253" length="9"/>
<text>influenza</text>
</annotation>
<annotation id="534">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="6263" length="2"/>
<text>PA</text>
</annotation>
<annotation id="555">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="6266" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="125">
<infon key="score">0.99884176</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6271" length="11"/>
<text>active site</text>
</annotation>
<annotation id="126">
<infon key="score">0.9987612</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6354" length="2"/>
<text>19</text>
</annotation>
<annotation id="127">
<infon key="score">0.99912053</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6358" length="3"/>
<text>H2L</text>
</annotation>
<annotation id="128">
<infon key="score">0.9987998</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6371" length="4"/>
<text>Mg2+</text>
</annotation>
<annotation id="129">
<infon key="score">0.9989767</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6476" length="13"/>
<text>triethylamine</text>
</annotation>
<annotation id="130">
<infon key="score">0.9799634</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6534" length="12"/>
<text>Mg(HL)2∙4H2O</text>
</annotation>
<annotation id="131">
<infon key="score">0.98885256</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6630" length="7"/>
<text>d6-DMSO</text>
</annotation>
<annotation id="132">
<infon key="score">0.9973355</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6691" length="6"/>
<text>1H-NMR</text>
</annotation>
<annotation id="133">
<infon key="score">0.9893941</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6698" length="8"/>
<text>spectrum</text>
</annotation>
<annotation id="134">
<infon key="score">0.9976766</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6784" length="7"/>
<text>13C-NMR</text>
</annotation>
<annotation id="135">
<infon key="score">0.898486</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6792" length="8"/>
<text>spectrum</text>
</annotation>
<annotation id="136">
<infon key="score">0.6472191</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7002" length="2"/>
<text>IR</text>
</annotation>
<annotation id="137">
<infon key="score">0.91154146</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7005" length="8"/>
<text>spectrum</text>
</annotation>
<annotation id="138">
<infon key="score">0.9450806</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7172" length="8"/>
<text>ESI-mass</text>
</annotation>
<annotation id="139">
<infon key="score">0.7451172</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:44:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7181" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="140">
<infon key="score">0.9956361</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7193" length="18"/>
<text>elemental analysis</text>
</annotation>
<annotation id="141">
<infon key="score">0.96558464</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:59:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7234" length="12"/>
<text>Mg(HL)2∙4H2O</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>7274</offset>
<text>The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1). The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups. By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm. The opposite trend is observed in the range 300–350 nm, where an isosbestic point is present close to 335 nm. When the same experiment was performed with 23, a different behavior was observed. Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear. Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum. These results will be revisited during the discussion of the biological activity.</text>
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<infon key="score">0.9965074</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7302" length="15"/>
<text>N-acylhydrazone</text>
</annotation>
<annotation id="143">
<infon key="score">0.9987508</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:52:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7334" length="9"/>
<text>magnesium</text>
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<annotation id="144">
<infon key="score">0.9987264</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7385" length="23"/>
<text>UV-visible spectroscopy</text>
</annotation>
<annotation id="145">
<infon key="score">0.99818856</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7410" length="21"/>
<text>UV-visible titrations</text>
</annotation>
<annotation id="146">
<infon key="score">0.99549353</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7435" length="2"/>
<text>23</text>
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<annotation id="147">
<infon key="score">0.9924136</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7442" length="2"/>
<text>19</text>
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<annotation id="148">
<infon key="score">0.50728977</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7450" length="17"/>
<text>increasing amount</text>
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<annotation id="149">
<infon key="score">0.99768364</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7471" length="11"/>
<text>Mg(CH3COO)2</text>
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<annotation id="623">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<location offset="7512" length="8"/>
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<annotation id="150">
<infon key="score">0.99344337</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:45Z</infon>
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<location offset="7524" length="2"/>
<text>19</text>
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<annotation id="151">
<infon key="score">0.9985274</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7651" length="11"/>
<text>Mg(CH3COO)2</text>
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<annotation id="152">
<infon key="score">0.9965898</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7907" length="2"/>
<text>23</text>
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<annotation id="153">
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7974" length="5"/>
<text>Mg2+,</text>
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<annotation id="154">
<infon key="score">0.99524873</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8134" length="2"/>
<text>19</text>
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<annotation id="155">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8141" length="2"/>
<text>23</text>
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<annotation id="616">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:58Z</infon>
<location offset="8144" length="10"/>
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<annotation id="156">
<infon key="score">0.99888766</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:00:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8159" length="4"/>
<text>Mg2+</text>
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<infon key="score">0.99557614</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:01:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8188" length="2"/>
<text>19</text>
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<annotation id="158">
<infon key="score">0.9941742</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:01:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8290" length="2"/>
<text>23</text>
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<annotation id="625">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:01:14Z</infon>
<location offset="8420" length="2"/>
<text>UV</text>
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<annotation id="624">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:57:23Z</infon>
<location offset="8423" length="8"/>
<text>spectrum</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>8540</offset>
<text>Inhibition of the PA-Nter enzyme</text>
<annotation id="535">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="8558" length="2"/>
<text>PA</text>
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<annotation id="556">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="8561" length="4"/>
<text>Nter</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>8573</offset>
<text>All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays). The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.</text>
<annotation id="159">
<infon key="score">0.98925996</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8636" length="9"/>
<text>influenza</text>
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<annotation id="160">
<infon key="score">0.9990226</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8646" length="12"/>
<text>endonuclease</text>
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<annotation id="161">
<infon key="score">0.9988707</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:01:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8665" length="29"/>
<text>enzymatic plasmid-based assay</text>
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<annotation id="536">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="8712" length="2"/>
<text>PA</text>
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<annotation id="557">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="8715" length="4"/>
<text>Nter</text>
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<annotation id="626">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:01:59Z</infon>
<location offset="8735" length="28"/>
<text>cell-based influenza methods</text>
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<annotation id="627">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:10Z</infon>
<location offset="8770" length="42"/>
<text>virus yield and vRNP reconstitution assays</text>
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<annotation id="162">
<infon key="score">0.9907825</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8944" length="20"/>
<text>dose-response curves</text>
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<annotation id="163">
<infon key="score">0.99843997</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9006" length="2"/>
<text>10</text>
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<annotation id="164">
<infon key="score">0.9982728</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9010" length="2"/>
<text>13</text>
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<annotation id="165">
<infon key="score">0.99854183</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9017" length="2"/>
<text>23</text>
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<annotation id="628">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:41Z</infon>
<location offset="9035" length="38"/>
<text>PA-enzyme or vRNP reconstitution assay</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9075</offset>
<text>The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively). When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8). The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11). Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2). Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing. 11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4). Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.</text>
<annotation id="166">
<infon key="score">0.9975967</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9098" length="4"/>
<text>IC50</text>
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<annotation id="167">
<infon key="score">0.99913883</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9115" length="41"/>
<text>N’-2,3-dihydroxybenzylidene semicarbazide</text>
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<annotation id="168">
<infon key="score">0.8099994</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9158" length="1"/>
<text>1</text>
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<annotation id="635">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:51Z</infon>
<location offset="9245" length="1"/>
<text>3</text>
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<annotation id="169">
<infon key="score">0.78925484</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9320" length="1"/>
<text>5</text>
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<annotation id="170">
<infon key="score">0.7282984</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:28Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9326" length="1"/>
<text>7</text>
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<infon key="score">0.99741757</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9329" length="4"/>
<text>IC50</text>
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<annotation id="172">
<infon key="score">0.998898</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:13Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9404" length="24"/>
<text>2,3-dihydroxybenzylidene</text>
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<infon key="score">0.99892056</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:18Z</infon>
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<location offset="9463" length="30"/>
<text>2-hydroxy-3-methoxybenzylidene</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:03:39Z</infon>
<location offset="9532" length="1"/>
<text>2</text>
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<annotation id="632">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:03:55Z</infon>
<location offset="9535" length="1"/>
<text>4</text>
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<annotation id="633">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:06Z</infon>
<location offset="9538" length="1"/>
<text>6</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:15Z</infon>
<location offset="9544" length="1"/>
<text>8</text>
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<infon key="score">0.9979487</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9576" length="4"/>
<text>IC50</text>
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<infon key="score">0.6382894</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9651" length="1"/>
<text>7</text>
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<infon key="score">0.98685247</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:28Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9694" length="1"/>
<text>9</text>
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<annotation id="177">
<infon key="score">0.9808538</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:04:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9697" length="2"/>
<text>10</text>
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<infon key="score">0.9842451</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9704" length="2"/>
<text>11</text>
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<infon key="score">0.9821738</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9720" length="2"/>
<text>11</text>
</annotation>
<annotation id="180">
<infon key="score">0.9929395</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9802" length="2"/>
<text>27</text>
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<infon key="score">0.93515146</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9917" length="2"/>
<text>11</text>
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<infon key="score">0.9458555</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9948" length="2"/>
<text>11</text>
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<infon key="score">0.78353983</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10051" length="10"/>
<text>coordinate</text>
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<infon key="score">0.9987627</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:52Z</infon>
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<location offset="10183" length="17"/>
<text>crystal structure</text>
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="10237" length="2"/>
<text>PA</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="10240" length="4"/>
<text>Nter</text>
</annotation>
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<infon key="score">0.5075802</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
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<location offset="10245" length="12"/>
<text>endonuclease</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10258" length="15"/>
<text>in complex with</text>
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<infon key="score">0.9987085</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:53:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10288" length="4"/>
<text>EGCG</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10329</offset>
<text>The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series. The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM). When one OH was added at position 2 of the R1 ring (14), the activity was lost. Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM). The addition of a 3-methoxy group (19) abolished all inhibitory activity. This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4). Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature. Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds). This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra). At this point, change of the substituents in R1 represents the next logical step. Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively). When two or three OH groups are present in R1, their spatial disposition greatly affects the activity. In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently. The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5. The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.</text>
<annotation id="538">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="10333" length="2"/>
<text>PA</text>
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<annotation id="559">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="10336" length="4"/>
<text>Nter</text>
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<annotation id="636">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:06:47Z</infon>
<location offset="10508" length="5"/>
<text>13–23</text>
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<annotation id="188">
<infon key="score">0.68469006</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10680" length="2"/>
<text>13</text>
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<annotation id="189">
<infon key="score">0.99552864</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10707" length="4"/>
<text>IC50</text>
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<annotation id="190">
<infon key="score">0.9441393</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10774" length="2"/>
<text>14</text>
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<annotation id="191">
<infon key="score">0.98732704</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:40:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10885" length="2"/>
<text>15</text>
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<annotation id="192">
<infon key="score">0.98189247</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10889" length="4"/>
<text>IC50</text>
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<annotation id="193">
<infon key="score">0.9942561</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:00Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10931" length="2"/>
<text>18</text>
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<annotation id="194">
<infon key="score">0.99097943</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10935" length="4"/>
<text>IC50</text>
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<annotation id="195">
<infon key="score">0.9926069</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11009" length="2"/>
<text>17</text>
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<annotation id="196">
<infon key="score">0.98846984</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11013" length="4"/>
<text>IC50</text>
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<annotation id="197">
<infon key="score">0.99281037</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11064" length="2"/>
<text>19</text>
</annotation>
<annotation id="663">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:20Z</infon>
<location offset="11210" length="5"/>
<text>14–19</text>
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<annotation id="198">
<infon key="score">0.9937058</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11355" length="2"/>
<text>18</text>
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<annotation id="199">
<infon key="score">0.9915317</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:07:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11393" length="3"/>
<text>M2+</text>
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<annotation id="200">
<infon key="score">0.9978527</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11409" length="11"/>
<text>active site</text>
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<annotation id="201">
<infon key="score">0.996293</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11459" length="4"/>
<text>IC50</text>
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<annotation id="202">
<infon key="score">0.987648</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11507" length="2"/>
<text>15</text>
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<annotation id="203">
<infon key="score">0.99806106</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11751" length="11"/>
<text>active site</text>
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<annotation id="204">
<infon key="score">0.99656165</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11958" length="14"/>
<text>hydrogen bonds</text>
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<annotation id="205">
<infon key="score">0.99873257</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12008" length="30"/>
<text>molecular docking calculations</text>
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<annotation id="206">
<infon key="score">0.99835294</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12043" length="14"/>
<text>X-ray analysis</text>
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<annotation id="207">
<infon key="score">0.9963385</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12071" length="2"/>
<text>23</text>
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<annotation id="208">
<infon key="score">0.9927866</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12074" length="15"/>
<text>in complex with</text>
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<annotation id="539">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="12090" length="2"/>
<text>PA</text>
</annotation>
<annotation id="560">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="12093" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="209">
<infon key="score">0.98792946</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12228" length="2"/>
<text>15</text>
</annotation>
<annotation id="210">
<infon key="score">0.99426925</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12251" length="2"/>
<text>16</text>
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<annotation id="211">
<infon key="score">0.99376845</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12291" length="4"/>
<text>IC50</text>
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<annotation id="212">
<infon key="score">0.9953263</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12315" length="2"/>
<text>15</text>
</annotation>
<annotation id="213">
<infon key="score">0.9959229</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12322" length="2"/>
<text>16</text>
</annotation>
<annotation id="214">
<infon key="score">0.9899739</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:41:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12525" length="2"/>
<text>20</text>
</annotation>
<annotation id="215">
<infon key="score">0.98735577</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12529" length="2"/>
<text>21</text>
</annotation>
<annotation id="666">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:18Z</infon>
<location offset="12533" length="2"/>
<text>22</text>
</annotation>
<annotation id="667">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:25Z</infon>
<location offset="12540" length="2"/>
<text>23</text>
</annotation>
<annotation id="540">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="12565" length="2"/>
<text>PA</text>
</annotation>
<annotation id="561">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="12568" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="629">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<location offset="12600" length="4"/>
<text>IC50</text>
</annotation>
<annotation id="664">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:01Z</infon>
<location offset="12630" length="2"/>
<text>21</text>
</annotation>
<annotation id="665">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:09Z</infon>
<location offset="12637" length="2"/>
<text>23</text>
</annotation>
<annotation id="630">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<location offset="12641" length="4"/>
<text>IC50</text>
</annotation>
<annotation id="668">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:33Z</infon>
<location offset="12786" length="2"/>
<text>23</text>
</annotation>
<annotation id="541">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="12947" length="2"/>
<text>PA</text>
</annotation>
<annotation id="562">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="12950" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="669">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:41Z</infon>
<location offset="12985" length="2"/>
<text>14</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13044</offset>
<text>Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12. On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25). Still lower activity was seen with the pyridine analogue 24. Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.</text>
<annotation id="647">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:48Z</infon>
<location offset="13108" length="9"/>
<text>chelation</text>
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<annotation id="216">
<infon key="score">0.99587184</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13142" length="2"/>
<text>15</text>
</annotation>
<annotation id="217">
<infon key="score">0.99225724</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:42:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13198" length="2"/>
<text>12</text>
</annotation>
<annotation id="218">
<infon key="score">0.99825305</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13285" length="4"/>
<text>IC50</text>
</annotation>
<annotation id="219">
<infon key="score">0.9990565</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13340" length="30"/>
<text>3,4,5-trihydroxybenzohydrazide</text>
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<annotation id="220">
<infon key="score">0.9956102</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:07Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13371" length="2"/>
<text>28</text>
</annotation>
<annotation id="221">
<infon key="score">0.99730444</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13424" length="2"/>
<text>26</text>
</annotation>
<annotation id="222">
<infon key="score">0.9970535</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:12Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13447" length="2"/>
<text>25</text>
</annotation>
<annotation id="223">
<infon key="score">0.9971583</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13509" length="2"/>
<text>24</text>
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<annotation id="542">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="13615" length="2"/>
<text>PA</text>
</annotation>
<annotation id="563">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="13618" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="520">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<location offset="13623" length="12"/>
<text>endonuclease</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>13766</offset>
<text>Inhibition of vRNP activity or virus replication in cells</text>
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<infon key="score">0.9976355</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:10:07Z</infon>
<infon key="identifier">GO:</infon>
<location offset="13780" length="4"/>
<text>vRNP</text>
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<annotation id="225">
<infon key="score">0.9874249</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13797" length="5"/>
<text>virus</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13824</offset>
<text>To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3). For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay. This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors. Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety. As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.</text>
<annotation id="226">
<infon key="score">0.6290952</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13846" length="9"/>
<text>influenza</text>
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<annotation id="593">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="13856" length="5"/>
<text>virus</text>
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<annotation id="227">
<infon key="score">0.9537156</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13884" length="4"/>
<text>1–28</text>
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<annotation id="228">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:10:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13922" length="35"/>
<text>influenza vRNP reconstitution assay</text>
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<annotation id="229">
<infon key="score">0.99535996</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:10:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13961" length="5"/>
<text>human</text>
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<annotation id="230">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14049" length="4"/>
<text>EC50</text>
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<annotation id="231">
<infon key="score">0.9428053</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:10:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14069" length="17"/>
<text>virus yield assay</text>
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<annotation id="232">
<infon key="score">0.8899718</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14090" length="9"/>
<text>influenza</text>
</annotation>
<annotation id="594">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="14100" length="5"/>
<text>virus</text>
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<annotation id="233">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14185" length="15"/>
<text>N-acylhydrazone</text>
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<annotation id="234">
<infon key="score">0.9985456</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:10:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14267" length="25"/>
<text>vRNP reconstitution assay</text>
</annotation>
<annotation id="235">
<infon key="score">0.9965383</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:47:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14339" length="5"/>
<text>viral</text>
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<annotation id="612">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:50:22Z</infon>
<location offset="14345" length="3"/>
<text>RNA</text>
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<annotation id="236">
<infon key="score">0.5546336</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14414" length="2"/>
<text>PA</text>
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<annotation id="237">
<infon key="score">0.9939942</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14440" length="4"/>
<text>EC50</text>
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<annotation id="238">
<infon key="score">0.7582195</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:44:46Z</infon>
<infon key="identifier">GO:</infon>
<location offset="14446" length="4"/>
<text>vRNP</text>
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<annotation id="239">
<infon key="score">0.9961493</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14455" length="4"/>
<text>EC90</text>
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<annotation id="595">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="14461" length="5"/>
<text>virus</text>
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<annotation id="240">
<infon key="score">0.9972523</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14528" length="2"/>
<text>15</text>
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<annotation id="241">
<infon key="score">0.9963448</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14535" length="5"/>
<text>20–23</text>
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<annotation id="242">
<infon key="score">0.9966973</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14614" length="2"/>
<text>15</text>
</annotation>
<annotation id="243">
<infon key="score">0.9963566</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14628" length="2"/>
<text>20</text>
</annotation>
<annotation id="244">
<infon key="score">0.9705081</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14631" length="2"/>
<text>23</text>
</annotation>
<annotation id="637">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:30Z</infon>
<location offset="14685" length="24"/>
<text>enzymatic PA-Nter assays</text>
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<annotation id="245">
<infon key="score">0.9964953</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14763" length="2"/>
<text>21</text>
</annotation>
<annotation id="246">
<infon key="score">0.9962042</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14767" length="2"/>
<text>22</text>
</annotation>
<annotation id="247">
<infon key="score">0.9944267</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14774" length="2"/>
<text>23</text>
</annotation>
<annotation id="248">
<infon key="score">0.9941497</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:11:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14861" length="1"/>
<text>9</text>
</annotation>
<annotation id="249">
<infon key="score">0.9900468</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14864" length="2"/>
<text>10</text>
</annotation>
<annotation id="250">
<infon key="score">0.99025047</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14871" length="2"/>
<text>11</text>
</annotation>
<annotation id="251">
<infon key="score">0.9884703</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14876" length="2"/>
<text>10</text>
</annotation>
<annotation id="252">
<infon key="score">0.9921761</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14883" length="2"/>
<text>22</text>
</annotation>
<annotation id="253">
<infon key="score">0.996451</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14914" length="4"/>
<text>EC50</text>
</annotation>
<annotation id="254">
<infon key="score">0.99858147</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14926" length="25"/>
<text>vRNP reconstitution assay</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>14990</offset>
<text>The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23). Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis. However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.</text>
<annotation id="255">
<infon key="score">0.9966097</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:16Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14994" length="9"/>
<text>hydrazide</text>
</annotation>
<annotation id="256">
<infon key="score">0.9948773</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:12:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15004" length="2"/>
<text>28</text>
</annotation>
<annotation id="596">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="15023" length="5"/>
<text>virus</text>
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<annotation id="638">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:04Z</infon>
<location offset="15049" length="19"/>
<text>vRNP reconstitution</text>
</annotation>
<annotation id="257">
<infon key="score">0.9985487</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15166" length="2"/>
<text>18</text>
</annotation>
<annotation id="258">
<infon key="score">0.9972357</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15173" length="5"/>
<text>21–23</text>
</annotation>
<annotation id="259">
<infon key="score">0.9956599</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15302" length="2"/>
<text>28</text>
</annotation>
<annotation id="260">
<infon key="score">0.99789184</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15343" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="261">
<infon key="score">0.9966673</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15535" length="4"/>
<text>EC50</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15701</offset>
<text>Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8. Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.</text>
<annotation id="262">
<infon key="score">0.99871427</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15733" length="24"/>
<text>2,3-dihydroxybenzylidene</text>
</annotation>
<annotation id="263">
<infon key="score">0.9609433</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15764" length="1"/>
<text>3</text>
</annotation>
<annotation id="264">
<infon key="score">0.9444579</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15767" length="1"/>
<text>5</text>
</annotation>
<annotation id="265">
<infon key="score">0.9343455</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15773" length="1"/>
<text>7</text>
</annotation>
<annotation id="266">
<infon key="score">0.9988452</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:13:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15779" length="30"/>
<text>2-hydroxy-3-methoxybenzylidene</text>
</annotation>
<annotation id="267">
<infon key="score">0.9820941</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15823" length="1"/>
<text>4</text>
</annotation>
<annotation id="268">
<infon key="score">0.9549013</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15826" length="1"/>
<text>6</text>
</annotation>
<annotation id="269">
<infon key="score">0.97834396</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15832" length="1"/>
<text>8</text>
</annotation>
<annotation id="270">
<infon key="score">0.9932685</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15878" length="10"/>
<text>vRNP assay</text>
</annotation>
<annotation id="271">
<infon key="score">0.9910517</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15895" length="4"/>
<text>CC50</text>
</annotation>
<annotation id="272">
<infon key="score">0.95631367</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15925" length="17"/>
<text>selectivity index</text>
</annotation>
<annotation id="273">
<infon key="score">0.98919183</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15953" length="4"/>
<text>CC50</text>
</annotation>
<annotation id="274">
<infon key="score">0.9908751</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15961" length="4"/>
<text>EC50</text>
</annotation>
<annotation id="275">
<infon key="score">0.99630535</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16003" length="2"/>
<text>18</text>
</annotation>
<annotation id="276">
<infon key="score">0.99594265</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16010" length="2"/>
<text>19</text>
</annotation>
<annotation id="277">
<infon key="score">0.998738</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:13Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16027" length="24"/>
<text>2,3-dihydroxybenzylidene</text>
</annotation>
<annotation id="278">
<infon key="score">0.9988715</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16055" length="30"/>
<text>2-hydroxy-3-methoxybenzylidene</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>16192</offset>
<text>Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively). For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay. On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28. Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay. This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner. To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.</text>
<annotation id="279">
<infon key="score">0.9978528</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16197" length="15"/>
<text>N-acylhydrazone</text>
</annotation>
<annotation id="280">
<infon key="score">0.99858165</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:55:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16254" length="15"/>
<text>enzymatic assay</text>
</annotation>
<annotation id="281">
<infon key="score">0.9975471</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16330" length="2"/>
<text>14</text>
</annotation>
<annotation id="282">
<infon key="score">0.99714905</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16337" length="2"/>
<text>19</text>
</annotation>
<annotation id="283">
<infon key="score">0.99172443</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16348" length="4"/>
<text>EC50</text>
</annotation>
<annotation id="284">
<infon key="score">0.9981059</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16433" length="1"/>
<text>9</text>
</annotation>
<annotation id="285">
<infon key="score">0.9972927</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16436" length="2"/>
<text>11</text>
</annotation>
<annotation id="286">
<infon key="score">0.9962166</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:16Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16440" length="2"/>
<text>13</text>
</annotation>
<annotation id="287">
<infon key="score">0.9952614</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:18Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16444" length="5"/>
<text>15–21</text>
</annotation>
<annotation id="288">
<infon key="score">0.9978259</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16451" length="2"/>
<text>23</text>
</annotation>
<annotation id="289">
<infon key="score">0.99684983</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16455" length="2"/>
<text>24</text>
</annotation>
<annotation id="290">
<infon key="score">0.9975376</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16462" length="2"/>
<text>26</text>
</annotation>
<annotation id="641">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:43Z</infon>
<infon key="error:misaligned:641">16505->16506</infon>
<location offset="16506" length="17"/>
<text>cell-based assays</text>
</annotation>
<annotation id="291">
<infon key="score">0.9952685</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16535" length="4"/>
<text>EC50</text>
</annotation>
<annotation id="292">
<infon key="score">0.99638575</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16563" length="10"/>
<text>vRNP assay</text>
</annotation>
<annotation id="293">
<infon key="score">0.9971117</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16613" length="4"/>
<text>EC90</text>
</annotation>
<annotation id="294">
<infon key="score">0.9901541</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16632" length="17"/>
<text>virus yield assay</text>
</annotation>
<annotation id="295">
<infon key="score">0.9980354</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16709" length="1"/>
<text>7</text>
</annotation>
<annotation id="296">
<infon key="score">0.9968274</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16712" length="2"/>
<text>10</text>
</annotation>
<annotation id="297">
<infon key="score">0.99701536</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16716" length="2"/>
<text>14</text>
</annotation>
<annotation id="298">
<infon key="score">0.9983103</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16720" length="2"/>
<text>22</text>
</annotation>
<annotation id="299">
<infon key="score">0.9979874</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16724" length="2"/>
<text>25</text>
</annotation>
<annotation id="300">
<infon key="score">0.9980386</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16731" length="2"/>
<text>28</text>
</annotation>
<annotation id="301">
<infon key="score">0.99742246</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16740" length="15"/>
<text>N-acylhydrazone</text>
</annotation>
<annotation id="302">
<infon key="score">0.99691087</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:15:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16806" length="10"/>
<text>vRNP assay</text>
</annotation>
<annotation id="303">
<infon key="score">0.99795216</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16823" length="2"/>
<text>14</text>
</annotation>
<annotation id="304">
<infon key="score">0.9975484</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16830" length="2"/>
<text>19</text>
</annotation>
<annotation id="305">
<infon key="score">0.9897635</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16841" length="4"/>
<text>EC50</text>
</annotation>
<annotation id="306">
<infon key="score">0.9986216</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:55:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16922" length="15"/>
<text>enzymatic assay</text>
</annotation>
<annotation id="307">
<infon key="score">0.9979644</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:47:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16991" length="5"/>
<text>viral</text>
</annotation>
<annotation id="308">
<infon key="score">0.99281305</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:49:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16997" length="10"/>
<text>polymerase</text>
</annotation>
<annotation id="521">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<location offset="17014" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="309">
<infon key="score">0.99821377</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17108" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="310">
<infon key="score">0.9980268</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17257" length="5"/>
<text>virus</text>
</annotation>
<annotation id="311">
<infon key="score">0.98708135</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:49:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17270" length="10"/>
<text>polymerase</text>
</annotation>
<annotation id="613">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:50:22Z</infon>
<location offset="17291" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="312">
<infon key="score">0.9981158</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17355" length="5"/>
<text>virus</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>17388</offset>
<text>Docking studies</text>
<annotation id="313">
<infon key="score">0.9958949</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17388" length="15"/>
<text>Docking studies</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17404</offset>
<text>In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG. Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands. It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.</text>
<annotation id="314">
<infon key="score">0.9987568</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17480" length="19"/>
<text>docking simulations</text>
</annotation>
<annotation id="315">
<infon key="score">0.99794</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:16:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17503" length="12"/>
<text>GOLD program</text>
</annotation>
<annotation id="543">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="17591" length="2"/>
<text>PA</text>
</annotation>
<annotation id="564">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="17594" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="316">
<infon key="score">0.91759837</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17599" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="317">
<infon key="score">0.99875635</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17612" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="318">
<infon key="score">0.99836403</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:53:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17628" length="4"/>
<text>EGCG</text>
</annotation>
<annotation id="319">
<infon key="score">0.9987803</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17762" length="12"/>
<text>superimposed</text>
</annotation>
<annotation id="320">
<infon key="score">0.9953639</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17780" length="16"/>
<text>X-ray structures</text>
</annotation>
<annotation id="544">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="17818" length="2"/>
<text>PA</text>
</annotation>
<annotation id="565">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="17821" length="4"/>
<text>Nter</text>
</annotation>
<annotation id="321">
<infon key="score">0.74848473</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17826" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="322">
<infon key="score">0.9995722</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17915" length="5"/>
<text>Tyr24</text>
</annotation>
<annotation id="323">
<infon key="score">0.60865927</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18010" length="8"/>
<text>flexible</text>
</annotation>
<annotation id="643">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:43Z</infon>
<location offset="18038" length="17"/>
<text>docking procedure</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18057</offset>
<text>First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure. Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).</text>
<annotation id="324">
<infon key="score">0.99885863</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18064" length="25"/>
<text>test docking calculations</text>
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<infon key="score">0.99857724</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:53:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18097" length="4"/>
<text>EGCG</text>
</annotation>
<annotation id="326">
<infon key="score">0.99856424</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:18Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18103" length="9"/>
<text>L-742,001</text>
</annotation>
<annotation id="327">
<infon key="score">0.9986336</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18117" length="59"/>
<text>2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one</text>
</annotation>
<annotation id="328">
<infon key="score">0.97238517</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18269" length="17"/>
<text>docking procedure</text>
</annotation>
<annotation id="329">
<infon key="score">0.9985305</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18362" length="4"/>
<text>rmsd</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18410</offset>
<text>Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).</text>
<annotation id="330">
<infon key="score">0.99864346</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18416" length="7"/>
<text>docking</text>
</annotation>
<annotation id="331">
<infon key="score">0.9985702</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18435" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
<annotation id="332">
<infon key="score">0.99903184</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18551" length="18"/>
<text>active site cavity</text>
</annotation>
<annotation id="333">
<infon key="score">0.9172839</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18577" length="2"/>
<text>PA</text>
</annotation>
<annotation id="334">
<infon key="score">0.9988042</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18580" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="335">
<infon key="score">0.9974936</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:44:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18639" length="24"/>
<text>crystallographic studies</text>
</annotation>
<annotation id="336">
<infon key="score">0.9961355</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18728" length="3"/>
<text>M2+</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18777</offset>
<text>Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23. These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively). In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5). In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side. Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.</text>
<annotation id="650">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:00Z</infon>
<location offset="18836" length="19"/>
<text>GOLD cluster docked</text>
</annotation>
<annotation id="337">
<infon key="score">0.99759704</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18879" length="2"/>
<text>23</text>
</annotation>
<annotation id="338">
<infon key="score">0.53585047</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18901" length="10"/>
<text>structures</text>
</annotation>
<annotation id="339">
<infon key="score">0.99429953</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19019" length="2"/>
<text>23</text>
</annotation>
<annotation id="340">
<infon key="score">0.98400265</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19038" length="10"/>
<text>coordinate</text>
</annotation>
<annotation id="341">
<infon key="score">0.99596596</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19057" length="3"/>
<text>M2+</text>
</annotation>
<annotation id="342">
<infon key="score">0.9987978</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19073" length="11"/>
<text>active site</text>
</annotation>
<annotation id="343">
<infon key="score">0.99744594</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:12Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19147" length="2"/>
<text>23</text>
</annotation>
<annotation id="344">
<infon key="score">0.99637127</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19176" length="29"/>
<text>hydrogen bonding interactions</text>
</annotation>
<annotation id="345">
<infon key="score">0.9284351</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19221" length="9"/>
<text>catalytic</text>
</annotation>
<annotation id="346">
<infon key="score">0.9994398</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19231" length="6"/>
<text>Lys134</text>
</annotation>
<annotation id="347">
<infon key="score">0.99946314</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19258" length="5"/>
<text>Glu26</text>
</annotation>
<annotation id="348">
<infon key="score">0.99564654</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19334" length="2"/>
<text>23</text>
</annotation>
<annotation id="349">
<infon key="score">0.9964912</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19343" length="16"/>
<text>π–π interactions</text>
</annotation>
<annotation id="350">
<infon key="score">0.9994823</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19386" length="5"/>
<text>Tyr24</text>
</annotation>
<annotation id="351">
<infon key="score">0.81873834</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19442" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="352">
<infon key="score">0.9990146</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:53:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19472" length="4"/>
<text>EGCG</text>
</annotation>
<annotation id="353">
<infon key="score">0.9989192</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:18:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19481" length="9"/>
<text>L-742,001</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>19496</offset>
<text>The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety. The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134. Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate. Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108. In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).</text>
<annotation id="354">
<infon key="score">0.9544191</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19538" length="2"/>
<text>15</text>
</annotation>
<annotation id="355">
<infon key="score">0.83134425</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:44:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19550" length="13"/>
<text>fitness value</text>
</annotation>
<annotation id="356">
<infon key="score">0.9965567</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19695" length="29"/>
<text>hydrogen bonding interactions</text>
</annotation>
<annotation id="357">
<infon key="score">0.9994832</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19730" length="6"/>
<text>Tyr130</text>
</annotation>
<annotation id="358">
<infon key="score">0.99628747</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19750" length="20"/>
<text>cation–π interaction</text>
</annotation>
<annotation id="359">
<infon key="score">0.99945813</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19776" length="6"/>
<text>Lys134</text>
</annotation>
<annotation id="360">
<infon key="score">0.99948764</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19784" length="6"/>
<text>Tyr130</text>
</annotation>
<annotation id="361">
<infon key="score">0.99882525</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19801" length="6"/>
<text>pocket</text>
</annotation>
<annotation id="362">
<infon key="score">0.99945635</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19827" length="6"/>
<text>Arg124</text>
</annotation>
<annotation id="363">
<infon key="score">0.9987018</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:50:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19904" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="364">
<infon key="score">0.94156957</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19928" length="2"/>
<text>15</text>
</annotation>
<annotation id="365">
<infon key="score">0.9967854</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19961" length="29"/>
<text>hydrogen bonding interactions</text>
</annotation>
<annotation id="366">
<infon key="score">0.9993685</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20023" length="5"/>
<text>Arg82</text>
</annotation>
<annotation id="367">
<infon key="score">0.9993944</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20033" length="6"/>
<text>Asp108</text>
</annotation>
<annotation id="368">
<infon key="score">0.8217899</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20055" length="9"/>
<text>chelation</text>
</annotation>
<annotation id="369">
<infon key="score">0.98686737</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:00Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20076" length="3"/>
<text>M2+</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>20152</offset>
<text>It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro. The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.</text>
<annotation id="370">
<infon key="score">0.9976574</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20196" length="2"/>
<text>23</text>
</annotation>
<annotation id="371">
<infon key="score">0.99680054</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20203" length="2"/>
<text>15</text>
</annotation>
<annotation id="372">
<infon key="score">0.9988625</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:21:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20315" length="16"/>
<text>highly conserved</text>
</annotation>
<annotation id="373">
<infon key="score">0.999537</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:17:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20344" length="5"/>
<text>Tyr24</text>
</annotation>
<annotation id="374">
<infon key="score">0.99951553</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20351" length="5"/>
<text>Glu26</text>
</annotation>
<annotation id="375">
<infon key="score">0.9995223</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20358" length="6"/>
<text>Arg124</text>
</annotation>
<annotation id="376">
<infon key="score">0.99954116</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20366" length="6"/>
<text>Tyr130</text>
</annotation>
<annotation id="377">
<infon key="score">0.99951816</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:19:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20377" length="6"/>
<text>Lys134</text>
</annotation>
<annotation id="378">
<infon key="score">0.9048646</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20418" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="379">
<infon key="score">0.9975988</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20559" length="2"/>
<text>15</text>
</annotation>
<annotation id="380">
<infon key="score">0.9984628</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20563" length="4"/>
<text>IC50</text>
</annotation>
<annotation id="381">
<infon key="score">0.997436</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:20:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20582" length="2"/>
<text>19</text>
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<annotation id="382">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:51:59Z</infon>
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<location offset="20648" length="10"/>
<text>coordinate</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>20915</offset>
<text>Crystallographic Studies</text>
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<infon key="updated_at">2023-09-20T16:21:22Z</infon>
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<location offset="20915" length="24"/>
<text>Crystallographic Studies</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>20940</offset>
<text>Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess. While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed. Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor. As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night. This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA). The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7). The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B). The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain. It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7). These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.</text>
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<infon key="updated_at">2023-09-20T16:21:18Z</infon>
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<location offset="20962" length="14"/>
<text>co-crystallize</text>
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<infon key="updated_at">2023-09-20T15:42:01Z</infon>
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<text>PA</text>
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<text>Nter</text>
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<infon key="updated_at">2023-09-20T16:22:17Z</infon>
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<text>15</text>
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<infon key="updated_at">2023-09-20T16:22:20Z</infon>
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<text>20</text>
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<annotation id="387">
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<infon key="updated_at">2023-09-20T16:22:22Z</infon>
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<text>21</text>
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<infon key="updated_at">2023-09-20T16:22:25Z</infon>
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<text>23</text>
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<infon key="updated_at">2023-09-20T16:21:38Z</infon>
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<text>crystals</text>
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<annotation id="390">
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<infon key="updated_at">2023-09-20T16:22:12Z</infon>
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<location offset="21122" length="16"/>
<text>electron density</text>
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<infon key="score">0.9992843</infon>
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<infon key="updated_at">2023-09-20T16:21:32Z</infon>
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<text>apo</text>
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<annotation id="392">
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<infon key="updated_at">2023-09-20T16:21:39Z</infon>
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<infon key="updated_at">2023-09-20T16:22:12Z</infon>
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<location offset="21301" length="16"/>
<text>electron density</text>
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<infon key="score">0.9993243</infon>
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<infon key="updated_at">2023-09-20T16:21:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21438" length="3"/>
<text>apo</text>
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<annotation id="395">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:21:39Z</infon>
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<location offset="21442" length="8"/>
<text>crystals</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:22:27Z</infon>
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<location offset="21516" length="2"/>
<text>23</text>
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<infon key="updated_at">2023-09-20T16:21:39Z</infon>
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<location offset="21524" length="8"/>
<text>crystals</text>
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<annotation id="398">
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<infon key="updated_at">2023-09-20T16:44:58Z</infon>
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<location offset="21617" length="9"/>
<text>structure</text>
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<annotation id="399">
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<infon key="updated_at">2023-09-20T16:22:12Z</infon>
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<location offset="21645" length="16"/>
<text>electron density</text>
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<infon key="score">0.71468544</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:21:43Z</infon>
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<location offset="21707" length="17"/>
<text>complex structure</text>
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<annotation id="401">
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<infon key="updated_at">2023-09-20T16:44:14Z</infon>
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<location offset="21780" length="19"/>
<text>docking simulations</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:52:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21851" length="9"/>
<text>manganese</text>
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<infon key="score">0.999305</infon>
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<infon key="updated_at">2023-09-20T16:17:13Z</infon>
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<location offset="21919" length="5"/>
<text>Tyr24</text>
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<infon key="updated_at">2023-09-20T16:08:11Z</infon>
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<location offset="22021" length="14"/>
<text>hydrogen bonds</text>
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<infon key="score">0.999268</infon>
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<infon key="updated_at">2023-09-20T16:19:34Z</infon>
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<location offset="22059" length="5"/>
<text>Glu26</text>
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<infon key="updated_at">2023-09-20T16:35:34Z</infon>
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<text>Lys34</text>
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<infon key="updated_at">2023-09-20T16:19:34Z</infon>
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<location offset="22232" length="5"/>
<text>Glu26</text>
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<infon key="score">0.9991905</infon>
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<infon key="updated_at">2023-09-20T16:35:34Z</infon>
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<location offset="22242" length="5"/>
<text>Lys34</text>
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<passage>
<infon key="section_type">CONCL</infon>
<infon key="type">title_1</infon>
<offset>22335</offset>
<text>Conclusions</text>
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<passage>
<infon key="section_type">CONCL</infon>
<infon key="type">paragraph</infon>
<offset>22347</offset>
<text>The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest. The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells). The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.</text>
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<infon key="updated_at">2023-09-20T15:43:43Z</infon>
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<location offset="22398" length="9"/>
<text>influenza</text>
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<annotation id="522">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<location offset="22463" length="12"/>
<text>endonuclease</text>
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<infon key="updated_at">2023-09-20T15:43:43Z</infon>
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<location offset="22488" length="9"/>
<text>influenza</text>
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<infon key="score">0.785363</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:09Z</infon>
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<location offset="22498" length="28"/>
<text>RNA-dependent RNA polymerase</text>
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<infon key="score">0.999037</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
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<location offset="22620" length="15"/>
<text>N-acylhydrazone</text>
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<infon key="score">0.97529185</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:22Z</infon>
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<location offset="22719" length="4"/>
<text>EC90</text>
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<infon key="updated_at">2023-09-20T16:15:59Z</infon>
<location offset="22732" length="17"/>
<text>virus yield assay</text>
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<infon key="updated_at">2023-09-20T15:43:43Z</infon>
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<location offset="22753" length="9"/>
<text>influenza</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="22763" length="5"/>
<text>virus</text>
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<infon key="score">0.99737966</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:12Z</infon>
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<location offset="22795" length="9"/>
<text>structure</text>
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<infon key="score">0.99909306</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22812" length="15"/>
<text>N-acylhydrazone</text>
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<infon key="score">0.99650013</infon>
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<infon key="updated_at">2023-09-20T16:23:23Z</infon>
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<text>23</text>
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<infon key="updated_at">2023-09-20T16:44:18Z</infon>
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<location offset="22831" length="15"/>
<text>co-crystallized</text>
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<infon key="identifier">PR:</infon>
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<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<location offset="22852" length="2"/>
<text>PA</text>
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<location offset="22855" length="4"/>
<text>Nter</text>
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<infon key="updated_at">2023-09-20T16:43:49Z</infon>
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<location offset="22943" length="11"/>
<text>coordinates</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22959" length="5"/>
<text>metal</text>
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22977" length="11"/>
<text>active site</text>
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<annotation id="523">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<location offset="23184" length="12"/>
<text>endonuclease</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23209" length="9"/>
<text>influenza</text>
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<annotation id="423">
<infon key="score">0.89355963</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23219" length="28"/>
<text>RNA-dependent RNA polymerase</text>
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<annotation id="424">
<infon key="score">0.99830073</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23287" length="18"/>
<text>carbonic anhydrase</text>
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<infon key="score">0.9986998</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:23:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23307" length="19"/>
<text>histone deacetylase</text>
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<infon key="score">0.8409801</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:52:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23332" length="5"/>
<text>HIV-1</text>
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<infon key="score">0.9830672</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:52:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23338" length="9"/>
<text>integrase</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:43:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23352" length="5"/>
<text>metal</text>
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</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>23412</offset>
<text>Experimental Section</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>23433</offset>
<text>Materials and methods. Chemistry</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>23466</offset>
<text>All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the compounds was determined by elemental analysis and verified to be ≥95% for all synthesized molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The attenuate total reflectance IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000–400 cm−1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means of a direct exposure probe mounting on the tip of a Re-filament with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer. UV–Vis spectra were recorded on an Evolution 260 Bio Thermo spectrophotometer by using cells of 1 cm path length. UV-vis absorption spectra of 19 and 23 were registered using a ca. 10−5 M solution in methanol. Each metal/ligand system was studied by titrating a 2.8 ml sample of the ligand solution with a methanolic solution of Mg(CH3COO)2; 8–12 spectra of samples with M:L molar ratio ranging from 0 to 6 were measured.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>24870</offset>
<text>Synthesis of the ligands (general procedure)</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>24915</offset>
<text>All the N-acylhydrazones were prepared in a manner similar to reported procedures. Briefly, to a solution of the aldehyde in absolute ethanol or toluene, an equimolar amount of the hydrazide dissolved in the same solvent was added. The mixture was refluxed for 6 hours, cooled at room temperature and concentrated in vacuum. The resulting precipitate was filtered off, washed with cold ethanol and dried in vacuum.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>25330</offset>
<text>3,4,5-trihydroxybenzohydrazide (28) and 3,4,5-trimethoxybenzohydrazide (29) were obtained by reaction of the corresponding methyl esters with hydrazine monohydrate. Hydrazine was added to an ethanol suspension of the ester and stirred at room temperature until the solute completely dissolved. Reaction mixture was then refluxed overnight. On concentrating the solution, a precipitate was observed, which was filtered and washed with cold ethanol. Chemical characterization of 1–29 and of Mg(HL)2 4H2O is collected in the Supplementary Information.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>25881</offset>
<text>Computational Studies</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>25903</offset>
<text>The crystal structure of PA-Nter endonuclease in complex with EGCG was retrieved from the RCSB Protein Data Bank (entry code 4AWM). The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5. The charge on the metal ions was set as +2. EGCG, L-742,001, and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one structures were extracted from their X-ray complexes (PDB IDs 4AWM, 4W9S and 4E5H respectively). The other ligand structures were constructed using Discovery Studio 2.5.5 (Accelrys, Discovery Studio) and energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient methods.</text>
<annotation id="655">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:37:10Z</infon>
<location offset="26050" length="5"/>
<text>water</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>26618</offset>
<text>The minimized ligands were docked in their corresponding proteins by means of GOLD Suite 5.0.1. The region of interest used by the GOLD program was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side-chain of residue Tyr24 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore was chosen as fitness function. The standard default settings were used in all calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom rmsd, were clustered together. The best GOLD-calculated conformation was used both for analysis and representation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>27401</offset>
<text>Plasmid-based endonuclease assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>27434</offset>
<text>This enzymatic assay was performed according to a previously published method. One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2. The reaction was incubated at 37 °C for 2 h and then stopped by heat inactivation (80 °C, 20 min), followed by visualization of the endonucleolytic digestion of the plasmid by gel electrophoresis on a 1% agarose gel with ethidium bromide staining. The amount of remaining intact plasmid was quantified by ImageQuant TL software (GE Healthcare, Diegem, Belgium). The percentage inhibition of PA endonuclease activity was plotted against the compound concentration on a semi-logarithmic plot, using GraphPad Prism software (GraphPad Software, La Jolla, CA). The 50% inhibitory concentrations (IC50) were obtained by nonlinear least-squares regression analysis of the results from three independent experiments. 2,4-Dioxo-4-phenylbutanoic acid (DPBA; Interchim, Montluçon, France) was included as the reference compound.</text>
<annotation id="661">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:37Z</infon>
<location offset="27746" length="11"/>
<text>presence of</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>28740</offset>
<text>Cells and media</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>28756</offset>
<text>MDCK cells (a kind gift from M. Matrosovich, Marburg, Germany) and HEK293T cells (purchased from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate. During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2 mM L-glutamine, and 2 μg/ml tosyl phenylalanyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO). The cells were incubated in a humidified atmosphere containing 5% CO2.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>29417</offset>
<text>vRNP reconstitution assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>29443</offset>
<text>The procedure to determine the inhibitory effect of the compounds on influenza virus vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere. Briefly, the four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the fluc reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity. EC50 was defined as the compound concentration causing 50% reduction in the vRNP-driven firefly luciferase signal, as compared to cells receiving medium instead of compound. These EC50 values were calculated by interpolation assuming a semi-log dose-response effect using GraphPad Prism software. In parallel, compound cytotoxic activity was determined in untransfected HEK293T cells which had been incubated with serial dilutions of the compounds for 24 h, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega). These spectrophotometric data were used to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by 50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) was included as the reference compound.</text>
<annotation id="662">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:37Z</infon>
<location offset="29894" length="11"/>
<text>presence of</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>31011</offset>
<text>Virus yield assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>31029</offset>
<text>We previously published in full detail the virus yield assay to determine the anti-influenza virus activity in MDCK cell cultures. Briefly, one day prior to infection, MDCK cells were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h incubation at 35 °C, the virus amount in the supernatants was estimated by determining the viral genome copy number in a one-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay (CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe. The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments. In parallel, the CC50 values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as the reference compound.</text>
<annotation id="657">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:12Z</infon>
<location offset="31774" length="4"/>
<text>EC99</text>
</annotation>
<annotation id="659">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:22Z</infon>
<location offset="31795" length="4"/>
<text>EC90</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32233</offset>
<text>Crystallographic analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32259</offset>
<text>A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies. The details of cloning, over-expression and purification are described elsewhere. Briefly, the gene was cloned into pET52b vector and transformed into BL21 (DE3) cells, and the protein was expressed in LB medium overnight at 18 °C after induction at an OD600 ~0.8 with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG). The protein was purified from cell lysates by HisTrap affinity chromatography and the 10xHis tag was removed by digestion with thrombin. The protein was further purified by gel filtration using a Superdex 75 size-exclusion chromatography column in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM TCEP. The protein was concentrated to 10–12 mg/ml for crystallization. Crystals were grown in 0.2 M MgCl2, 2 mM MnCl2, 0.1 M Tris pH 8.5, 30% (w/v) PEG 4000 using the hanging drop method. For determination of the protein-inhibitor complex structure, the powder of the inhibitor was sprinkled on a 2 μl drop of a 1:1 ratio mixture of protein solution and well solution, on a cover slide hanging over 500 μl well solution, and left overnight. Next day, the crystals were cryo-protected using well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. The data were collected at the 22-ID beam line maintained by Southeast Regional Collaborative Access Team (SERCAT) at the Advanced Photon Source, Argonne National Laboratory. The data were indexed, integrated and scaled using the HKL2000 suite of programs. Phase determination, structure refinement and model building were completed using Phaser, Refmac and Coot (part of the CCP4 package). The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement. The details of the data collection and refinement statistics are given in Table S1.</text>
<annotation id="651">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:35:15Z</infon>
<location offset="32276" length="8"/>
<text>PANΔLoop</text>
</annotation>
<annotation id="652">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:35:15Z</infon>
<location offset="34023" length="8"/>
<text>PANΔLoop</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>34198</offset>
<text>Additional Information</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34221</offset>
<text>How to cite this article: Carcelli, M. et al. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes. Sci. Rep. 6, 31500; doi: 10.1038/srep31500 (2016).</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>34416</offset>
<text>Supplementary Material</text>
</passage>
<passage>
<infon key="fpage">1487</infon>
<infon key="lpage">1531</infon>
<infon key="name_0">surname:Lamb;given-names:R. A.</infon>
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<offset>39272</offset>
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<text>Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor</text>
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<infon key="name_0">surname:Otwinowski;given-names:Z.</infon>
<infon key="name_1">surname:Minor;given-names:W.</infon>
<infon key="name_2">surname:Sweet;given-names:R. M.</infon>
<infon key="section_type">REF</infon>
<infon key="source">In Methods in Enzymology, Volume 276: Macromolecular Crystallography, part A</infon>
<infon key="type">ref</infon>
<infon key="year">1997</infon>
<offset>39752</offset>
<text>Processing of X-ray diffraction data collected in oscillation mode</text>
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<passage>
<infon key="fpage">235</infon>
<infon key="lpage">42</infon>
<infon key="name_0">surname:Winn;given-names:M. D.</infon>
<infon key="pub-id_pmid">21460441</infon>
<infon key="section_type">REF</infon>
<infon key="source">Acta Crystallogr. D Biol Crystallogr</infon>
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<text>Overview of the CCP4 suite and current developments</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>39871</offset>
<text>Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.C., D.R., A.G. and M.S. drug design and chemical synthesis; L.D.L. docking studies; G.K. and S.W.W. crystallographic studies; A.S. and L.N. biological studies.</text>
</passage>
<passage>
<infon key="file">srep31500-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>40191</offset>
<text>Chemical structures of some prototype inhibitors of influenza virus endonuclease.</text>
<annotation id="578">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:43:44Z</infon>
<location offset="40243" length="9"/>
<text>influenza</text>
</annotation>
<annotation id="598">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="40253" length="5"/>
<text>virus</text>
</annotation>
<annotation id="429">
<infon key="score">0.9990734</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40259" length="12"/>
<text>endonuclease</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>40273</offset>
<text>Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..</text>
<annotation id="430">
<infon key="score">0.9933729</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:32:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40295" length="16"/>
<text>enzymatic assays</text>
</annotation>
<annotation id="431">
<infon key="score">0.9974349</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="40313" length="4"/>
<text>IC50</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>40370</offset>
<text>General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A). Chemical structures of compounds 1–27 (B).</text>
<annotation id="432">
<infon key="score">0.9967518</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40392" length="16"/>
<text>N-acylhydrazones</text>
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<annotation id="648">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:16Z</infon>
<location offset="40409" length="4"/>
<text>1–27</text>
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<annotation id="433">
<infon key="score">0.9969893</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40418" length="10"/>
<text>hydrazides</text>
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<annotation id="434">
<infon key="score">0.9928181</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40429" length="2"/>
<text>28</text>
</annotation>
<annotation id="435">
<infon key="score">0.9833402</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40436" length="2"/>
<text>29</text>
</annotation>
<annotation id="649">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:44Z</infon>
<location offset="40477" length="4"/>
<text>1–27</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>40491</offset>
<text>Overview of the structure-activity relationship for compounds 1–27.</text>
<annotation id="436">
<infon key="score">0.5852117</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:33:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40553" length="4"/>
<text>1–27</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f4.jpg</infon>
<infon key="id">f4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>40561</offset>
<text>Scheme of possible binding modes of the studied N-acylhydrazones.</text>
<annotation id="437">
<infon key="score">0.9985623</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:46Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40609" length="16"/>
<text>N-acylhydrazones</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f5.jpg</infon>
<infon key="id">f5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>40627</offset>
<text>First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
2011,
51, 2778]. Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres. Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text>
<annotation id="438">
<infon key="score">0.99032193</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40652" length="19"/>
<text>GOLD cluster docked</text>
</annotation>
<annotation id="439">
<infon key="score">0.9923509</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="40694" length="2"/>
<text>23</text>
</annotation>
<annotation id="583">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<location offset="40729" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="440">
<infon key="score">0.84009457</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="40745" length="2"/>
<text>PA</text>
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<annotation id="441">
<infon key="score">0.99712664</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40748" length="12"/>
<text>endonuclease</text>
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<annotation id="442">
<infon key="score">0.99829596</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="40782" length="6"/>
<text>pocket</text>
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<annotation id="443">
<infon key="score">0.99809974</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40843" length="7"/>
<text>LIGPLUS</text>
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<annotation id="444">
<infon key="score">0.9962585</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="40949" length="14"/>
<text>Hydrogen bonds</text>
</annotation>
<annotation id="445">
<infon key="score">0.9576666</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41127" length="19"/>
<text>GOLD cluster docked</text>
</annotation>
<annotation id="446">
<infon key="score">0.99771047</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41173" length="7"/>
<text>LIGPLUS</text>
</annotation>
<annotation id="447">
<infon key="score">0.99637365</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41199" length="14"/>
<text>hydrogen bonds</text>
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<annotation id="448">
<infon key="score">0.9970145</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41256" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f6.jpg</infon>
<infon key="id">f6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>41286</offset>
<text>(A) Binding mode of compound 15 (orange) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
2011,
51, 2778]. Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres. (B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text>
<annotation id="449">
<infon key="score">0.984543</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="41315" length="2"/>
<text>15</text>
</annotation>
<annotation id="450">
<infon key="score">0.99838465</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="41327" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="451">
<infon key="score">0.5495376</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="41343" length="2"/>
<text>PA</text>
</annotation>
<annotation id="452">
<infon key="score">0.9897317</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41346" length="12"/>
<text>endonuclease</text>
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<annotation id="453">
<infon key="score">0.99815685</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="41380" length="6"/>
<text>pocket</text>
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<annotation id="454">
<infon key="score">0.9978351</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41441" length="7"/>
<text>LIGPLUS</text>
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<annotation id="455">
<infon key="score">0.9961376</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41547" length="14"/>
<text>Hydrogen bonds</text>
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<annotation id="456">
<infon key="score">0.92023325</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="41707" length="2"/>
<text>15</text>
</annotation>
<annotation id="457">
<infon key="score">0.9973815</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41726" length="7"/>
<text>LIGPLUS</text>
</annotation>
<annotation id="458">
<infon key="score">0.9955679</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:08:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41752" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="459">
<infon key="score">0.9968715</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:34:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41809" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f7.jpg</infon>
<infon key="id">f7</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>41839</offset>
<text>Crystal structure of PANΔLoop in complex with compound 23.</text>
<annotation id="460">
<infon key="score">0.9985323</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:44:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="41839" length="17"/>
<text>Crystal structure</text>
</annotation>
<annotation id="461">
<infon key="score">0.9988675</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:35:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="41860" length="8"/>
<text>PANΔLoop</text>
</annotation>
<annotation id="462">
<infon key="score">0.99797577</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:45:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="41869" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="463">
<infon key="score">0.6296788</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:35:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="41894" length="2"/>
<text>23</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31500-f7.jpg</infon>
<infon key="id">f7</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>41902</offset>
<text>Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres. Compound 23 is shown in sticks with yellow carbons. Protein secondary structure is shown as ribbons in salmon color. 2Fo-Fc electron density map contoured at 1σ is shown as blue mesh. Hydrogen bonds and metal coordination are shown with dotted lines. The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively. The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.</text>
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<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
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<location offset="42501" length="5"/>
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<location offset="42686" length="6"/>
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<location offset="42719" length="17"/>
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<infon key="updated_at">2023-09-20T16:37:04Z</infon>
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<infon key="updated_at">2023-09-20T15:55:31Z</infon>
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<infon key="annotator">cleaner0</infon>
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<text>Nter</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:41:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42929" length="12"/>
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<annotation id="654">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:36:59Z</infon>
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<text>cellular influenza virus assays</text>
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<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
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<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th rowspan="3" align="left" valign="top" charoff="50">Compound</th><th rowspan="2" align="center" valign="top" charoff="50">Enzyme assay with PA-Nter<xref ref-type="fn" rid="t1-fn1">a</xref></th><th colspan="4" align="center" valign="top" charoff="50">Virus yield assay in influenza virus-infected MDCK cells<xref ref-type="fn" rid="t1-fn2">b</xref></th><th colspan="2" align="center" valign="top" charoff="50">vRNP reconstitution assay in HEK293T cells<xref ref-type="fn" rid="t1-fn3">c</xref></th></tr><tr><th colspan="2" align="center" valign="top" charoff="50">Antiviral activity</th><th align="center" valign="top" charoff="50">Cytotoxicity</th><th rowspan="2" align="center" valign="top" charoff="50">SI<xref ref-type="fn" rid="t1-fn4">d</xref></th><th align="center" valign="top" charoff="50">Activity</th><th align="center" valign="top" charoff="50">Cytotoxicity</th></tr><tr><th align="center" valign="top" charoff="50">IC<sub>50</sub></th><th align="center" valign="top" charoff="50">EC<sub>99</sub></th><th align="center" valign="top" charoff="50">EC<sub>90</sub></th><th align="center" valign="top" charoff="50">CC<sub>50</sub></th><th align="center" valign="top" charoff="50">EC<sub>50</sub></th><th align="center" valign="top" charoff="50">CC<sub>50</sub></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>(1)</bold></td><td align="center" valign="top" charoff="50">24</td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn6">f</xref></td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">107</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(2)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">&gt;100</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(3)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">5.9</td><td align="center" valign="top" charoff="50">48</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(4)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">6.3</td><td align="center" valign="top" charoff="50">33</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(5)</bold></td><td align="center" valign="top" charoff="50">67</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">≥146</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">2.6</td><td align="center" valign="top" charoff="50">10</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(6)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;50</td><td align="center" valign="top" charoff="50">&gt;50</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">15</td><td align="center" valign="top" charoff="50">14</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(7)</bold></td><td align="center" valign="top" charoff="50">54</td><td align="center" valign="top" charoff="50">172</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.0</td><td align="center" valign="top" charoff="50">3.2</td><td align="center" valign="top" charoff="50">8.9</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(8)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">1.9</td><td align="center" valign="top" charoff="50">15</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(9)</bold></td><td align="center" valign="top" charoff="50">34</td><td align="center" valign="top" charoff="50">16</td><td align="center" valign="top" charoff="50">5.3</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;38</td><td align="center" valign="top" charoff="50">5.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(10)</bold></td><td align="center" valign="top" charoff="50">68</td><td align="center" valign="top" charoff="50">14</td><td align="center" valign="top" charoff="50">8.5</td><td align="center" valign="top" charoff="50">111</td><td align="center" valign="top" charoff="50">&gt;13</td><td align="center" valign="top" charoff="50">0.40</td><td align="center" valign="top" charoff="50">132</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(11)</bold></td><td align="center" valign="top" charoff="50">45</td><td align="center" valign="top" charoff="50">30</td><td align="center" valign="top" charoff="50">12</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;17</td><td align="center" valign="top" charoff="50">5.6</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(12)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">20</td><td align="center" valign="top" charoff="50">39</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(13)</bold></td><td align="center" valign="top" charoff="50">69</td><td align="center" valign="top" charoff="50">71</td><td align="center" valign="top" charoff="50">34</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;5.9</td><td align="center" valign="top" charoff="50">6.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(14)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">63</td><td align="center" valign="top" charoff="50">37</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;5.4</td><td align="center" valign="top" charoff="50">2.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(15)</bold></td><td align="center" valign="top" charoff="50">8.9</td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">7.5</td><td align="center" valign="top" charoff="50">≥172</td><td align="center" valign="top" charoff="50">≥23</td><td align="center" valign="top" charoff="50">14</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(16)</bold></td><td align="center" valign="top" charoff="50">454</td><td align="center" valign="top" charoff="50">67</td><td align="center" valign="top" charoff="50">28</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.1</td><td align="center" valign="top" charoff="50">5.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(17)</bold></td><td align="center" valign="top" charoff="50">482</td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">8.1</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">7.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(18)</bold></td><td align="center" valign="top" charoff="50">83</td><td align="center" valign="top" charoff="50">6.2</td><td align="center" valign="top" charoff="50">2.2</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;91</td><td align="center" valign="top" charoff="50">3.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(19)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">53</td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.7</td><td align="center" valign="top" charoff="50">5.7</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(20)</bold></td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">35</td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;18</td><td align="center" valign="top" charoff="50">2.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(21)</bold></td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">8.3</td><td align="center" valign="top" charoff="50">3.6</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;56</td><td align="center" valign="top" charoff="50">2.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(22)</bold></td><td align="center" valign="top" charoff="50">75</td><td align="center" valign="top" charoff="50">7.4</td><td align="center" valign="top" charoff="50">3.4</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;59</td><td align="center" valign="top" charoff="50">0.42</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(23)</bold></td><td align="center" valign="top" charoff="50">8.7</td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">3.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;57</td><td align="center" valign="top" charoff="50">3.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(24)</bold></td><td align="center" valign="top" charoff="50">131</td><td align="center" valign="top" charoff="50">58</td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.7</td><td align="center" valign="top" charoff="50">25</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(25)</bold></td><td align="center" valign="top" charoff="50">40</td><td align="center" valign="top" charoff="50">132</td><td align="center" valign="top" charoff="50">70</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.9</td><td align="center" valign="top" charoff="50">4.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(26)</bold></td><td align="center" valign="top" charoff="50">30</td><td align="center" valign="top" charoff="50">36</td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;15</td><td align="center" valign="top" charoff="50">5.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(27)</bold></td><td align="center" valign="top" charoff="50">36</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(28)</bold></td><td align="center" valign="top" charoff="50">40</td><td align="center" valign="top" charoff="50">158</td><td align="center" valign="top" charoff="50">85</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.4</td><td align="center" valign="top" charoff="50">7.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50">DPBA<xref ref-type="fn" rid="t1-fn5">e</xref></td><td align="center" valign="top" charoff="50">5.3</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td></tr><tr><td align="left" valign="top" charoff="50">Ribavirin</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">8.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;24</td><td align="center" valign="top" charoff="50">9.4</td><td align="center" valign="top" charoff="50">&gt;200</td></tr></tbody></table>
</infon>
<offset>42984</offset>
<text>Compound Enzyme assay with PA-Ntera Virus yield assay in influenza virus-infected MDCK cellsb vRNP reconstitution assay in HEK293T cellsc Antiviral activity Cytotoxicity SId Activity Cytotoxicity IC50 EC99 EC90 CC50 EC50 CC50 (1) 24 NDf ND ND 107 >200 (2) >500 ND ND ND >100 >200 (3) >500 ND ND >200 5.9 48 (4) >500 ND ND >200 6.3 33 (5) 67 >25 >25 ≥146 2.6 10 (6) >500 >50 >50 >200 15 14 (7) 54 172 100 >200 >2.0 3.2 8.9 (8) >500 >12.5 >12.5 >200 1.9 15 (9) 34 16 5.3 >200 >38 5.5 >200 (10) 68 14 8.5 111 >13 0.40 132 (11) 45 30 12 >200 >17 5.6 >200 (12) >500 >12.5 >12.5 >200 20 39 (13) 69 71 34 >200 >5.9 6.3 >200 (14) >500 63 37 >200 >5.4 2.3 >200 (15) 8.9 18 7.5 ≥172 ≥23 14 >200 (16) 454 67 28 >200 >7.1 5.2 >200 (17) 482 21 8.1 >200 >25 7.1 >200 (18) 83 6.2 2.2 >200 >91 3.3 >200 (19) >500 53 26 >200 >7.7 5.7 >200 (20) 18 35 11 >200 >18 2.2 >200 (21) 13 8.3 3.6 >200 >56 2.5 >200 (22) 75 7.4 3.4 >200 >59 0.42 >200 (23) 8.7 11 3.5 >200 >57 3.1 >200 (24) 131 58 26 >200 >7.7 25 >200 (25) 40 132 70 >200 >2.9 4.1 >200 (26) 30 36 13 >200 >15 5.5 >200 (27) 36 ND ND ND 21 >200 (28) 40 158 85 >200 >2.4 7.2 >200 DPBAe 5.3 ND ND ND ND ND Ribavirin ND 13 8.5 >200 >24 9.4 >200 </text>
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<infon key="updated_at">2023-09-20T16:44:22Z</infon>
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<infon key="updated_at">2023-09-20T16:15:59Z</infon>
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<infon key="updated_at">2023-09-20T16:44:26Z</infon>
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<infon key="updated_at">2023-09-20T16:02:48Z</infon>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<text>EC90</text>
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<annotation id="639">
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<location offset="43199" length="4"/>
<text>CC50</text>
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<annotation id="620">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="43204" length="4"/>
<text>EC50</text>
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<annotation id="640">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<text>CC50</text>
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<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
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<text>aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds. Cleavage of the substrate was assessed after 2 hr incubation. The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.</text>
<annotation id="489">
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<infon key="updated_at">2023-09-20T15:42:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44278" length="2"/>
<text>PA</text>
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<annotation id="569">
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<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-20T15:42:28Z</infon>
<location offset="44281" length="4"/>
<text>Nter</text>
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<infon key="updated_at">2023-09-20T16:38:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44290" length="9"/>
<text>incubated</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="44309" length="5"/>
<text>ssDNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="44336" length="4"/>
<text>Mn2+</text>
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<annotation id="493">
<infon key="score">0.9971878</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:02:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44445" length="4"/>
<text>IC50</text>
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<annotation id="494">
<infon key="score">0.99887514</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44557" length="43"/>
<text>nonlinear least-squares regression analysis</text>
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</passage>
<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>44687</offset>
<text>bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments. The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).</text>
<annotation id="660">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:14Z</infon>
<location offset="44718" length="11"/>
<text>influenza A</text>
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<annotation id="495">
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<infon key="type">taxonomy_domain</infon>
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<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44730" length="5"/>
<text>virus</text>
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<annotation id="601">
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="44805" length="5"/>
<text>virus</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44852" length="14"/>
<text>real-time qPCR</text>
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<annotation id="497">
<infon key="score">0.9965879</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44872" length="4"/>
<text>EC99</text>
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<infon key="score">0.9963899</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44881" length="4"/>
<text>EC90</text>
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<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:46:16Z</infon>
<location offset="44983" length="5"/>
<text>virus</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45127" length="4"/>
<text>CC50</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45188" length="24"/>
<text>MTS cell viability assay</text>
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<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>45226</offset>
<text>cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds. The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software). The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.</text>
<annotation id="501">
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<infon key="updated_at">2023-09-20T16:39:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45246" length="14"/>
<text>co-transfected</text>
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<infon key="updated_at">2023-09-20T16:39:31Z</infon>
<infon key="identifier">GO:</infon>
<location offset="45275" length="4"/>
<text>vRNP</text>
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45347" length="11"/>
<text>presence of</text>
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45383" length="4"/>
<text>EC50</text>
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<infon key="updated_at">2023-09-20T16:39:33Z</infon>
<infon key="identifier">GO:</infon>
<location offset="45461" length="4"/>
<text>vRNP</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:56:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45540" length="4"/>
<text>EC50</text>
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<infon key="updated_at">2023-09-20T16:44:29Z</infon>
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<location offset="45610" length="43"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45691" length="4"/>
<text>CC50</text>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45792" length="24"/>
<text>MTS cell viability assay</text>
</annotation>
</passage>
<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
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<text>dSI, selectivity index, defined as the ratio between the CC50 and EC90.</text>
<annotation id="510">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:39:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45824" length="3"/>
<text>dSI</text>
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45829" length="17"/>
<text>selectivity index</text>
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<infon key="score">0.99740654</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:14:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45881" length="4"/>
<text>CC50</text>
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<annotation id="513">
<infon key="score">0.9969945</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T16:38:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45890" length="4"/>
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<passage>
<infon key="file">t1.xml</infon>
<infon key="id">t1</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>45896</offset>
<text>eDPBA, 2,4-dioxo-4-phenylbutanoic acid.</text>
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<infon key="alt-title">Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</infon>
<infon key="article-id_doi">10.1371/journal.pone.0160694</infon>
<infon key="article-id_pmc">4981400</infon>
<infon key="article-id_pmid">27513867</infon>
<infon key="article-id_publisher-id">PONE-D-16-20928</infon>
<infon key="elocation-id">e0160694</infon>
<infon key="issue">8</infon>
<infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon>
<infon key="name_0">surname:Zhang;given-names:Yinglu</infon>
<infon key="name_1">surname:Rataj;given-names:Katarzyna</infon>
<infon key="name_2">surname:Simpson;given-names:Gordon G.</infon>
<infon key="name_3">surname:Tong;given-names:Liang</infon>
<infon key="name_4">surname:Candela;given-names:Hector</infon>
<infon key="name_5">surname:Tong;given-names:Liang</infon>
<infon key="name_6">surname:Tong;given-names:Liang</infon>
<infon key="name_7">surname:Simpson;given-names:Gordon G.</infon>
<infon key="name_8">surname:Simpson;given-names:Gordon G.</infon>
<infon key="notes">All relevant data are within the paper and its Supporting Information files.</infon>
<infon key="section_type">TITLE</infon>
<infon key="title">Data Availability</infon>
<infon key="type">front</infon>
<infon key="volume">11</infon>
<infon key="year">2016</infon>
<offset>0</offset>
<text>Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</text>
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<text>Crystal Structure</text>
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<text>SPOC</text>
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<text>Arabidopsis</text>
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<text>Flowering Regulator</text>
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<infon key="section_type">ABSTRACT</infon>
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<text>The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.</text>
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<text>FLOWERING LOCUS</text>
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<text>split ends</text>
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<text>RNA recognition motifs</text>
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<text>SPOC</text>
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<infon key="updated_at">2023-09-20T15:05:44Z</infon>
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<text>highly conserved</text>
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<infon key="type">protein</infon>
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<text>FPA</text>
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<infon key="updated_at">2023-09-20T12:51:58Z</infon>
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<text>plants</text>
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<text>SPEN</text>
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<infon key="updated_at">2023-09-20T12:48:36Z</infon>
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<text>crystal structure</text>
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<infon key="updated_at">2023-09-20T12:49:40Z</infon>
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<text>Arabidopsis thaliana</text>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<text>FPA</text>
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<text>SPOC</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:08Z</infon>
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<text>structure</text>
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<infon key="score">0.9994272</infon>
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<infon key="annotator">cleaner0</infon>
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<location offset="685" length="4"/>
<text>SPOC</text>
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<infon key="updated_at">2023-09-20T12:49:46Z</infon>
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<text>human</text>
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<text>SMRT/HDAC1 Associated Repressor Protein</text>
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<text>SHARP</text>
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<text>Structural and sequence analyses</text>
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<text>surface patch</text>
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<text>conserved</text>
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<text>plant</text>
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<text>FPA</text>
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<text>Mutations</text>
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<infon key="updated_at">2023-09-20T12:52:40Z</infon>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="998" length="3"/>
<text>FPA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
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<location offset="1040" length="4"/>
<text>SPOC</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1084" length="3"/>
<text>FPA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1102" length="3"/>
<text>RNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1147" length="3"/>
<text>FPA</text>
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<infon key="score">0.9993544</infon>
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<infon key="updated_at">2023-09-20T12:49:04Z</infon>
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<text>SPOC</text>
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<infon key="identifier">PR:</infon>
<location offset="1253" length="5"/>
<text>SHARP</text>
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<infon key="score">0.99941254</infon>
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<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1259" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">title_1</infon>
<offset>1277</offset>
<text>Introduction</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1290</offset>
<text>Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing.</text>
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<infon key="updated_at">2023-09-20T12:52:58Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>Eukaryotic</text>
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<infon key="identifier">CHEBI:</infon>
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<text>messenger RNAs</text>
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<infon key="identifier">CHEBI:</infon>
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<text>mRNAs</text>
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<infon key="updated_at">2023-09-20T12:53:07Z</infon>
<infon key="identifier">GO:</infon>
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<text>RNA polymerase II</text>
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<infon key="updated_at">2023-09-20T12:53:11Z</infon>
<infon key="identifier">GO:</infon>
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<text>Pol II</text>
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<infon key="updated_at">2023-09-20T14:43:32Z</infon>
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<text>eukaryotes</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1876</offset>
<text>Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.</text>
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<text>split ends</text>
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<infon key="updated_at">2023-09-20T12:50:59Z</infon>
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<location offset="1902" length="4"/>
<text>SPEN</text>
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<infon key="updated_at">2023-09-20T12:54:03Z</infon>
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<location offset="1945" length="20"/>
<text>RNA binding proteins</text>
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<infon key="score">0.99936414</infon>
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<infon key="updated_at">2023-09-20T12:51:19Z</infon>
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<location offset="2077" length="22"/>
<text>RNA recognition motifs</text>
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<annotation id="50">
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<infon key="updated_at">2023-09-20T12:51:24Z</infon>
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<text>RRMs</text>
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<infon key="updated_at">2023-09-20T12:54:01Z</infon>
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<text>conserved</text>
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<location offset="2123" length="36"/>
<text>SPEN paralog and ortholog C-terminal</text>
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<infon key="updated_at">2023-09-20T12:49:04Z</infon>
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<text>SPOC</text>
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<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
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<text>SPOC</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:50:59Z</infon>
<location offset="2284" length="4"/>
<text>SPEN</text>
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<passage>
<infon key="file">pone.0160694.g001.jpg</infon>
<infon key="id">pone.0160694.g001</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>2379</offset>
<text>Sequence conservation of SPOC domains.</text>
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<infon key="updated_at">2023-09-20T12:55:07Z</infon>
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<text>Sequence conservation</text>
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<text>SPOC</text>
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<passage>
<infon key="file">pone.0160694.g001.jpg</infon>
<infon key="id">pone.0160694.g001</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>2418</offset>
<text>(A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.</text>
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<infon key="score">0.9980342</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:54:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2446" length="11"/>
<text>A. thaliana</text>
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<annotation id="56">
<infon key="score">0.9989416</infon>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<text>FPA</text>
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<annotation id="57">
<infon key="score">0.9985844</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:04Z</infon>
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<location offset="2468" length="18"/>
<text>Sequence alignment</text>
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<text>SPOC</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2510" length="20"/>
<text>Arabidopsis thaliana</text>
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<infon key="score">0.9992167</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2531" length="3"/>
<text>FPA</text>
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<annotation id="60">
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<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2536" length="5"/>
<text>human</text>
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<annotation id="61">
<infon key="score">0.99920624</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:21Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2542" length="5"/>
<text>RBM15</text>
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<annotation id="62">
<infon key="score">0.7792917</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2549" length="10"/>
<text>Drosophila</text>
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<annotation id="63">
<infon key="score">0.9992182</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:50:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2560" length="4"/>
<text>SPEN</text>
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<annotation id="64">
<infon key="score">0.9206429</infon>
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<infon key="updated_at">2023-09-20T12:55:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2566" length="5"/>
<text>mouse</text>
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<annotation id="65">
<infon key="score">0.9989303</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:43:50Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2572" length="4"/>
<text>MINT</text>
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<annotation id="66">
<infon key="score">0.99851686</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2582" length="5"/>
<text>human</text>
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<annotation id="67">
<infon key="score">0.9991665</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2588" length="5"/>
<text>SHARP</text>
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<annotation id="68">
<infon key="score">0.93170136</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2607" length="15"/>
<text>surface patch 1</text>
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<annotation id="69">
<infon key="score">0.93146116</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2672" length="15"/>
<text>surface patch 2</text>
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<annotation id="70">
<infon key="score">0.9454224</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2749" length="9"/>
<text>structure</text>
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<annotation id="71">
<infon key="score">0.99068165</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2762" length="3"/>
<text>FPA</text>
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<annotation id="72">
<infon key="score">0.5914585</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2766" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="73">
<infon key="score">0.9987582</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2802" length="18"/>
<text>strictly conserved</text>
</annotation>
<annotation id="74">
<infon key="score">0.9979994</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2906" length="16"/>
<text>mostly conserved</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2931</offset>
<text>FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear.</text>
<annotation id="75">
<infon key="score">0.9988631</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2931" length="3"/>
<text>FPA</text>
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<annotation id="523">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:50:59Z</infon>
<location offset="2938" length="4"/>
<text>SPEN</text>
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<annotation id="76">
<infon key="score">0.9964304</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2961" length="20"/>
<text>Arabidopsis thaliana</text>
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<annotation id="77">
<infon key="score">0.9980305</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:51:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2992" length="6"/>
<text>plants</text>
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<annotation id="78">
<infon key="score">0.9736698</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3081" length="14"/>
<text>antisense RNAs</text>
</annotation>
<annotation id="79">
<infon key="score">0.5571705</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:36Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="3099" length="15"/>
<text>FLOWERING LOCUS</text>
</annotation>
<annotation id="80">
<infon key="score">0.5104294</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="3116" length="3"/>
<text>FLC</text>
</annotation>
<annotation id="81">
<infon key="score">0.9989016</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3150" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="82">
<infon key="score">0.54643095</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="3196" length="3"/>
<text>FLC</text>
</annotation>
<annotation id="83">
<infon key="score">0.9708284</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3200" length="14"/>
<text>antisense RNAs</text>
</annotation>
<annotation id="84">
<infon key="score">0.99701446</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3244" length="20"/>
<text>polyadenylation site</text>
</annotation>
<annotation id="85">
<infon key="score">0.97197783</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3290" length="19"/>
<text>histone demethylase</text>
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<annotation id="528">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<location offset="3342" length="3"/>
<text>FLC</text>
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</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3435</offset>
<text>Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information.</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="3446" length="4"/>
<text>SPOC</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:50:59Z</infon>
<location offset="3478" length="4"/>
<text>SPEN</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="3588" length="4"/>
<text>SPOC</text>
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<annotation id="86">
<infon key="score">0.9984439</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3604" length="11"/>
<text>A. thaliana</text>
</annotation>
<annotation id="87">
<infon key="score">0.9993006</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3616" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="88">
<infon key="score">0.9989404</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3624" length="5"/>
<text>human</text>
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<annotation id="89">
<infon key="score">0.995815</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:23Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3630" length="39"/>
<text>SMRT/HDAC1 Associated Repressor Protein</text>
</annotation>
<annotation id="90">
<infon key="score">0.9992428</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3671" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="91">
<infon key="score">0.99915516</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3715" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="491">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="3721" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3778</offset>
<text>As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs.</text>
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<infon key="score">0.9742827</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3904" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="93">
<infon key="score">0.9980663</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:48:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3932" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="94">
<infon key="score">0.99945563</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3957" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="95">
<infon key="score">0.99831086</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3972" length="11"/>
<text>A. thaliana</text>
</annotation>
<annotation id="96">
<infon key="score">0.9990339</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3984" length="3"/>
<text>FPA</text>
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<annotation id="97">
<infon key="score">0.9978776</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4021" length="9"/>
<text>structure</text>
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<annotation id="98">
<infon key="score">0.31347078</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4057" length="5"/>
<text>SHARP</text>
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<annotation id="492">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="4063" length="4"/>
<text>SPOC</text>
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<annotation id="99">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4157" length="9"/>
<text>structure</text>
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<annotation id="100">
<infon key="score">0.9984716</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
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<location offset="4177" length="13"/>
<text>surface patch</text>
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<annotation id="101">
<infon key="score">0.99805117</infon>
<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-20T12:57:32Z</infon>
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<location offset="4199" length="9"/>
<text>conserved</text>
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<location offset="4215" length="3"/>
<text>FPA</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>4232</offset>
<text>Results and Discussion</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>4255</offset>
<text>Structure of FPA SPOC domain</text>
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<infon key="score">0.9942719</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="4255" length="9"/>
<text>Structure</text>
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<annotation id="104">
<infon key="score">0.99878544</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4268" length="3"/>
<text>FPA</text>
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<annotation id="105">
<infon key="score">0.9995203</infon>
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<text>SPOC</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>4284</offset>
<text>The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF.</text>
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<text>crystal structure</text>
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<location offset="4313" length="4"/>
<text>SPOC</text>
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<infon key="identifier">MESH:</infon>
<location offset="4328" length="11"/>
<text>A. thaliana</text>
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<annotation id="109">
<infon key="score">0.99917847</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<location offset="4340" length="3"/>
<text>FPA</text>
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<annotation id="110">
<infon key="score">0.99822134</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4394" length="61"/>
<text>selenomethionyl single-wavelength anomalous dispersion method</text>
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<infon key="score">0.9976018</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4501" length="7"/>
<text>433–565</text>
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<annotation id="112">
<infon key="score">0.9992718</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4512" length="3"/>
<text>FPA</text>
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<annotation id="113">
<infon key="score">0.9976713</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:50Z</infon>
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<location offset="4535" length="7"/>
<text>439–460</text>
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<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4547" length="7"/>
<text>465–565</text>
</annotation>
<annotation id="115">
<infon key="score">0.7115964</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4574" length="7"/>
<text>crystal</text>
</annotation>
<annotation id="116">
<infon key="score">0.9846461</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4587" length="12"/>
<text>atomic model</text>
</annotation>
<annotation id="117">
<infon key="score">0.61089885</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:57:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4628" length="22"/>
<text>X-ray diffraction data</text>
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<annotation id="118">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4795" length="17"/>
<text>Ramachandran plot</text>
</annotation>
<annotation id="119">
<infon key="score">0.98965245</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4835" length="9"/>
<text>structure</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.t001.xml</infon>
<infon key="id">pone.0160694.t001</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_title_caption</infon>
<offset>4923</offset>
<text>Summary of crystallographic information.</text>
</passage>
<passage>
<infon key="file">pone.0160694.t001.xml</infon>
<infon key="id">pone.0160694.t001</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table</infon>
<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><tbody><tr><td align="left" rowspan="1" colspan="1">Resolution range (Å)<xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td><td align="center" rowspan="1" colspan="1">50–2.7 (2.8–2.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of observations</td><td align="center" rowspan="1" colspan="1">78,008</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub> (%)</td><td align="center" rowspan="1" colspan="1">10.5 (45.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">I/σI</td><td align="center" rowspan="1" colspan="1">24.1 (6.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Redundancy</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="center" rowspan="1" colspan="1">100 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic> factor (%)</td><td align="center" rowspan="1" colspan="1">19.2 (25.0)</td></tr><tr><td align="left" rowspan="1" colspan="1">Free <italic>R</italic> factor (%)</td><td align="center" rowspan="1" colspan="1">25.4 (35.4)</td></tr><tr><td align="left" rowspan="1" colspan="1">Rms deviation in bond lengths (Å)</td><td align="center" rowspan="1" colspan="1">0.017</td></tr><tr><td align="left" rowspan="1" colspan="1">Rms deviation in bond angles (°)</td><td align="center" rowspan="1" colspan="1">1.9</td></tr></tbody></table>
</infon>
<offset>4964</offset>
<text>Resolution range (Å)1 50–2.7 (2.8–2.7) Number of observations 78,008 Rmerge (%) 10.5 (45.3) I/σI 24.1 (6.3) Redundancy Completeness (%) 100 (100) R factor (%) 19.2 (25.0) Free R factor (%) 25.4 (35.4) Rms deviation in bond lengths (Å) 0.017 Rms deviation in bond angles (°) 1.9 </text>
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<infon key="score">0.99649537</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5123" length="8"/>
<text>R factor</text>
</annotation>
<annotation id="121">
<infon key="score">0.95946175</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5150" length="13"/>
<text>Free R factor</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.t001.xml</infon>
<infon key="id">pone.0160694.t001</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_footnote</infon>
<offset>5272</offset>
<text>1The numbers in parentheses are for the highest resolution shell.</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>5338</offset>
<text>The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.</text>
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<infon key="score">0.9988049</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:48:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5342" length="17"/>
<text>crystal structure</text>
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<annotation id="123">
<infon key="score">0.985938</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5367" length="3"/>
<text>FPA</text>
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<annotation id="493">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="5371" length="4"/>
<text>SPOC</text>
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<infon key="updated_at">2023-09-20T12:58:36Z</infon>
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<location offset="5394" length="45"/>
<text>seven-stranded, mostly anti-parallel β-barrel</text>
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<annotation id="125">
<infon key="score">0.9972434</infon>
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<infon key="updated_at">2023-09-20T12:58:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5441" length="5"/>
<text>β1-β7</text>
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<annotation id="126">
<infon key="score">0.9876231</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5458" length="7"/>
<text>helices</text>
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<annotation id="127">
<infon key="score">0.9962954</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5467" length="5"/>
<text>αA-αC</text>
</annotation>
<annotation id="128">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5512" length="7"/>
<text>strands</text>
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<annotation id="129">
<infon key="score">0.99939585</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5521" length="2"/>
<text>β1</text>
</annotation>
<annotation id="130">
<infon key="score">0.9993862</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5528" length="2"/>
<text>β3</text>
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<annotation id="131">
<infon key="score">0.9983072</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5560" length="5"/>
<text>Helix</text>
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<infon key="updated_at">2023-09-20T12:59:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5566" length="2"/>
<text>αB</text>
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<infon key="score">0.9992513</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5591" length="6"/>
<text>barrel</text>
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<annotation id="134">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5605" length="7"/>
<text>helices</text>
</annotation>
<annotation id="135">
<infon key="score">0.99948055</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5613" length="2"/>
<text>αA</text>
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<annotation id="136">
<infon key="score">0.99945194</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5620" length="2"/>
<text>αC</text>
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<annotation id="137">
<infon key="score">0.99912864</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5673" length="6"/>
<text>barrel</text>
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<annotation id="138">
<infon key="score">0.99915344</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5711" length="8"/>
<text>β-barrel</text>
</annotation>
<annotation id="139">
<infon key="score">0.9990128</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5738" length="4"/>
<text>loop</text>
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<annotation id="140">
<infon key="score">0.9920982</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5754" length="7"/>
<text>strands</text>
</annotation>
<annotation id="141">
<infon key="score">0.999331</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:42:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5762" length="2"/>
<text>β2</text>
</annotation>
<annotation id="142">
<infon key="score">0.9992975</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5769" length="2"/>
<text>β3</text>
</annotation>
<annotation id="143">
<infon key="score">0.9989806</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5792" length="10"/>
<text>disordered</text>
</annotation>
<annotation id="144">
<infon key="score">0.99722177</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5803" length="7"/>
<text>461–464</text>
</annotation>
<annotation id="145">
<infon key="score">0.99890804</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5838" length="6"/>
<text>barrel</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g002.jpg</infon>
<infon key="id">pone.0160694.g002</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>5966</offset>
<text>Crystal structure of the SPOC domain of A. thaliana FPA.</text>
<annotation id="146">
<infon key="score">0.99874413</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:48:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5966" length="17"/>
<text>Crystal structure</text>
</annotation>
<annotation id="494">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="5991" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="147">
<infon key="score">0.9980335</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6006" length="11"/>
<text>A. thaliana</text>
</annotation>
<annotation id="148">
<infon key="score">0.98107886</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6018" length="3"/>
<text>FPA</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g002.jpg</infon>
<infon key="id">pone.0160694.g002</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>6023</offset>
<text>(A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).</text>
<annotation id="149">
<infon key="score">0.9700408</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6053" length="9"/>
<text>structure</text>
</annotation>
<annotation id="150">
<infon key="score">0.984616</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6066" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="151">
<infon key="score">0.99939215</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6070" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="152">
<infon key="score">0.999288</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6178" length="8"/>
<text>β-barrel</text>
</annotation>
<annotation id="153">
<infon key="score">0.9964395</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:05:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6192" length="10"/>
<text>disordered</text>
</annotation>
<annotation id="154">
<infon key="score">0.9978326</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6221" length="7"/>
<text>460–465</text>
</annotation>
<annotation id="155">
<infon key="score">0.99287826</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6270" length="9"/>
<text>Structure</text>
</annotation>
<annotation id="156">
<infon key="score">0.99104744</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6287" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="157">
<infon key="score">0.9992855</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6291" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="158">
<infon key="score">0.9992698</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6331" length="8"/>
<text>β-barrel</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>6471</offset>
<text>Comparisons to structural homologs of the SPOC domain</text>
<annotation id="534">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:30Z</infon>
<location offset="6471" length="34"/>
<text>Comparisons to structural homologs</text>
</annotation>
<annotation id="495">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="6513" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>6525</offset>
<text>Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0.</text>
<annotation id="159">
<infon key="score">0.7332179</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6562" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="160">
<infon key="score">0.999361</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6566" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="161">
<infon key="score">0.9983363</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6623" length="15"/>
<text>DaliLite server</text>
</annotation>
<annotation id="162">
<infon key="score">0.9990138</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6660" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="163">
<infon key="score">0.99605185</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6672" length="9"/>
<text>structure</text>
</annotation>
<annotation id="164">
<infon key="score">0.9993979</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6723" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="165">
<infon key="score">0.9984281</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6738" length="5"/>
<text>human</text>
</annotation>
<annotation id="166">
<infon key="score">0.9990225</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6744" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="167">
<infon key="score">0.9980933</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:07:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6767" length="7"/>
<text>Z score</text>
</annotation>
<annotation id="530">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<location offset="6831" length="8"/>
<text>β-barrel</text>
</annotation>
<annotation id="168">
<infon key="score">0.9989506</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6863" length="4"/>
<text>Ku70</text>
</annotation>
<annotation id="169">
<infon key="score">0.9988921</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:08Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6872" length="4"/>
<text>Ku80</text>
</annotation>
<annotation id="170">
<infon key="score">0.997887</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6878" length="7"/>
<text>Z score</text>
</annotation>
<annotation id="171">
<infon key="score">0.9908906</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6918" length="20"/>
<text>chromodomain protein</text>
</annotation>
<annotation id="172">
<infon key="score">0.99925596</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:04Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6939" length="4"/>
<text>Chp1</text>
</annotation>
<annotation id="173">
<infon key="score">0.99778926</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6945" length="7"/>
<text>Z score</text>
</annotation>
<annotation id="174">
<infon key="score">0.99922377</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6977" length="28"/>
<text>activator interacting domain</text>
</annotation>
<annotation id="175">
<infon key="score">0.9984598</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7007" length="4"/>
<text>ACID</text>
</annotation>
<annotation id="176">
<infon key="score">0.9993298</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:15Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7020" length="5"/>
<text>Med25</text>
</annotation>
<annotation id="177">
<infon key="score">0.99764967</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7059" length="7"/>
<text>Z score</text>
</annotation>
<annotation id="178">
<infon key="score">0.99812615</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:07:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7116" length="7"/>
<text>Z score</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g003.jpg</infon>
<infon key="id">pone.0160694.g003</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>7135</offset>
<text>Structural homologs of the FPA SPOC domain.</text>
<annotation id="179">
<infon key="score">0.966769</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7162" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="496">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="7166" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g003.jpg</infon>
<infon key="id">pone.0160694.g003</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>7179</offset>
<text>(A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</text>
<annotation id="180">
<infon key="score">0.9266318</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7184" length="7"/>
<text>Overlay</text>
</annotation>
<annotation id="181">
<infon key="score">0.99402386</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7199" length="10"/>
<text>structures</text>
</annotation>
<annotation id="182">
<infon key="score">0.9952513</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7217" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="183">
<infon key="score">0.998982</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7221" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="184">
<infon key="score">0.9987124</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7248" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="497">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="7254" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="185">
<infon key="score">0.99656993</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7298" length="21"/>
<text>doubly-phosphorylated</text>
</annotation>
<annotation id="186">
<infon key="score">0.9844742</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7320" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="187">
<infon key="score">0.9982146</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:03:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7333" length="4"/>
<text>SMRT</text>
</annotation>
<annotation id="188">
<infon key="score">0.92117435</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7364" length="7"/>
<text>Overlay</text>
</annotation>
<annotation id="189">
<infon key="score">0.99422437</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7379" length="10"/>
<text>structures</text>
</annotation>
<annotation id="190">
<infon key="score">0.99556077</infon>
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<text>SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.</text>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<infon key="updated_at">2023-09-20T12:59:23Z</infon>
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<infon key="updated_at">2023-09-20T13:05:10Z</infon>
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<infon key="updated_at">2023-09-20T12:59:23Z</infon>
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<infon key="updated_at">2023-09-20T12:59:35Z</infon>
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<infon key="updated_at">2023-09-20T12:59:35Z</infon>
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<infon key="updated_at">2023-09-20T13:05:47Z</infon>
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<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-20T12:59:07Z</infon>
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<infon key="updated_at">2023-09-20T12:58:47Z</infon>
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<infon key="updated_at">2023-09-20T14:42:26Z</infon>
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<infon key="updated_at">2023-09-20T12:58:55Z</infon>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<infon key="updated_at">2023-09-20T13:06:45Z</infon>
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<text>peptide</text>
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<annotation id="240">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:05:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9102" length="10"/>
<text>β1-β2 loop</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9139</offset>
<text>The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.</text>
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<infon key="score">0.9979747</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:07:11Z</infon>
<infon key="identifier">GO:</infon>
<location offset="9143" length="9"/>
<text>Ku70-Ku80</text>
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<annotation id="543">
<infon key="type">oligomeric_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:22Z</infon>
<location offset="9153" length="12"/>
<text>hetero-dimer</text>
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<annotation id="532">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<location offset="9220" length="8"/>
<text>β-barrel</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:07:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9251" length="3"/>
<text>DNA</text>
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<annotation id="533">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<location offset="9277" length="8"/>
<text>β-barrel</text>
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<annotation id="243">
<infon key="score">0.9989766</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9297" length="4"/>
<text>Ku70</text>
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<annotation id="244">
<infon key="score">0.99886876</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:08Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9306" length="4"/>
<text>Ku80</text>
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<annotation id="544">
<infon key="type">oligomeric_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:22Z</infon>
<location offset="9318" length="12"/>
<text>hetero-dimer</text>
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<annotation id="245">
<infon key="score">0.9985916</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:05:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9375" length="5"/>
<text>loops</text>
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<annotation id="246">
<infon key="score">0.9872525</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:08:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9396" length="24"/>
<text>third and fourth strands</text>
</annotation>
<annotation id="247">
<infon key="score">0.99917185</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9428" length="6"/>
<text>barrel</text>
</annotation>
<annotation id="248">
<infon key="score">0.9990589</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:42:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9470" length="9"/>
<text>β-barrels</text>
</annotation>
<annotation id="249">
<infon key="score">0.9989037</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9489" length="17"/>
<text>DNA binding sites</text>
</annotation>
<annotation id="250">
<infon key="score">0.99702954</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9555" length="5"/>
<text>dsDNA</text>
</annotation>
<annotation id="549">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:08:26Z</infon>
<location offset="9577" length="11"/>
<text>long insert</text>
</annotation>
<annotation id="251">
<infon key="score">0.9117398</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9600" length="7"/>
<text>strands</text>
</annotation>
<annotation id="252">
<infon key="score">0.9974274</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:42:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9608" length="2"/>
<text>β2</text>
</annotation>
<annotation id="253">
<infon key="score">0.99721956</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9615" length="2"/>
<text>β3</text>
</annotation>
<annotation id="254">
<infon key="score">0.99237406</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:08:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9645" length="19"/>
<text>arch-like structure</text>
</annotation>
<annotation id="255">
<infon key="score">0.9949091</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="9681" length="5"/>
<text>dsDNA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9703</offset>
<text>Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).</text>
<annotation id="256">
<infon key="score">0.9983724</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9703" length="4"/>
<text>Chp1</text>
</annotation>
<annotation id="257">
<infon key="score">0.74675</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:08:55Z</infon>
<infon key="identifier">GO:</infon>
<location offset="9728" length="56"/>
<text>RNA-induced initiation of transcriptional gene silencing</text>
</annotation>
<annotation id="258">
<infon key="score">0.95602566</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:08:58Z</infon>
<infon key="identifier">GO:</infon>
<location offset="9786" length="4"/>
<text>RITS</text>
</annotation>
<annotation id="259">
<infon key="score">0.99872905</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9816" length="4"/>
<text>Chp1</text>
</annotation>
<annotation id="260">
<infon key="score">0.9989574</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:02Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9822" length="4"/>
<text>Tas3</text>
</annotation>
<annotation id="261">
<infon key="score">0.99926513</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9849" length="13"/>
<text>barrel domain</text>
</annotation>
<annotation id="262">
<infon key="score">0.9989255</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:42:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9871" length="13"/>
<text>second domain</text>
</annotation>
<annotation id="263">
<infon key="score">0.99880016</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9888" length="4"/>
<text>Chp1</text>
</annotation>
<annotation id="264">
<infon key="score">0.9988242</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9902" length="6"/>
<text>linker</text>
</annotation>
<annotation id="265">
<infon key="score">0.999301</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10013" length="8"/>
<text>β-barrel</text>
</annotation>
<annotation id="266">
<infon key="score">0.99884015</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10051" length="4"/>
<text>Tas3</text>
</annotation>
<annotation id="267">
<infon key="score">0.9993482</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10074" length="4"/>
<text>loop</text>
</annotation>
<annotation id="268">
<infon key="score">0.9989498</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10082" length="4"/>
<text>Tas3</text>
</annotation>
<annotation id="269">
<infon key="score">0.99536186</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10096" length="6"/>
<text>strand</text>
</annotation>
<annotation id="270">
<infon key="score">0.99474216</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10103" length="2"/>
<text>β3</text>
</annotation>
<annotation id="271">
<infon key="score">0.99932253</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10113" length="13"/>
<text>barrel domain</text>
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<annotation id="538">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:03:59Z</infon>
<location offset="10200" length="4"/>
<text>SMRT</text>
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<annotation id="547">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:45Z</infon>
<location offset="10205" length="7"/>
<text>peptide</text>
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<annotation id="272">
<infon key="score">0.9976284</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:10:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10213" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="273">
<infon key="score">0.4227668</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10229" length="5"/>
<text>SHARP</text>
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<annotation id="503">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="10235" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10263</offset>
<text>Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.</text>
<annotation id="274">
<infon key="score">0.6381052</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:43:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10263" length="8"/>
<text>Mediator</text>
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<annotation id="275">
<infon key="score">0.7621727</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:53:11Z</infon>
<infon key="identifier">GO:</infon>
<location offset="10328" length="6"/>
<text>Pol II</text>
</annotation>
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<infon key="score">0.99933845</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:01:16Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10340" length="5"/>
<text>Med25</text>
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<infon key="score">0.7525669</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10354" length="4"/>
<text>ACID</text>
</annotation>
<annotation id="278">
<infon key="score">0.99747354</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10397" length="4"/>
<text>VP16</text>
</annotation>
<annotation id="279">
<infon key="score">0.9693468</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:11:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10409" length="20"/>
<text>herpes simplex virus</text>
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<infon key="score">0.9971583</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10435" length="9"/>
<text>structure</text>
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<infon key="score">0.47994432</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:00:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10448" length="4"/>
<text>ACID</text>
</annotation>
<annotation id="282">
<infon key="score">0.98497874</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10464" length="5"/>
<text>helix</text>
</annotation>
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<infon key="score">0.99923664</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:05:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10511" length="10"/>
<text>β1-β2 loop</text>
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<annotation id="284">
<infon key="score">0.99889576</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:09:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10540" length="12"/>
<text>binding site</text>
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<annotation id="285">
<infon key="score">0.9980599</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10557" length="4"/>
<text>VP16</text>
</annotation>
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<infon key="score">0.99654675</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10598" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="287">
<infon key="score">0.9965989</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10618" length="7"/>
<text>strands</text>
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<infon key="score">0.998982</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10626" length="2"/>
<text>β1</text>
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<infon key="score">0.99914646</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10633" length="2"/>
<text>β3</text>
</annotation>
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<infon key="score">0.9992305</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10657" length="5"/>
<text>SHARP</text>
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<infon key="score">0.9992337</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:09:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10667" length="4"/>
<text>Tas3</text>
</annotation>
<annotation id="504">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="10672" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>10725</offset>
<text>A conserved surface patch in the FPA SPOC domain</text>
<annotation id="292">
<infon key="score">0.93878067</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:12:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10727" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="293">
<infon key="score">0.9084494</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10737" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="294">
<infon key="score">0.9349592</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10758" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="505">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="10762" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10774</offset>
<text>An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).</text>
<annotation id="506">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="10793" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="295">
<infon key="score">0.9878807</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10823" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="296">
<infon key="score">0.9886966</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10842" length="7"/>
<text>strands</text>
</annotation>
<annotation id="297">
<infon key="score">0.9989058</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10850" length="2"/>
<text>β1</text>
</annotation>
<annotation id="298">
<infon key="score">0.9990095</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10854" length="2"/>
<text>β3</text>
</annotation>
<annotation id="299">
<infon key="score">0.9991211</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10858" length="2"/>
<text>β5</text>
</annotation>
<annotation id="300">
<infon key="score">0.9990693</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10865" length="2"/>
<text>β6</text>
</annotation>
<annotation id="301">
<infon key="score">0.99898845</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:06:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10876" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="302">
<infon key="score">0.9987618</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10892" length="5"/>
<text>plant</text>
</annotation>
<annotation id="303">
<infon key="score">0.9980325</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10898" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="304">
<infon key="score">0.99669635</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10926" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="550">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:55Z</infon>
<location offset="10963" length="11"/>
<text>sub-patches</text>
</annotation>
<annotation id="305">
<infon key="score">0.9995813</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10990" length="6"/>
<text>Lys447</text>
</annotation>
<annotation id="306">
<infon key="score">0.9467047</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11001" length="6"/>
<text>strand</text>
</annotation>
<annotation id="307">
<infon key="score">0.99921155</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:13:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11008" length="2"/>
<text>β1</text>
</annotation>
<annotation id="308">
<infon key="score">0.9995802</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11013" length="6"/>
<text>Arg477</text>
</annotation>
<annotation id="309">
<infon key="score">0.99930096</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11021" length="2"/>
<text>β3</text>
</annotation>
<annotation id="310">
<infon key="score">0.9995828</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11026" length="6"/>
<text>Tyr515</text>
</annotation>
<annotation id="311">
<infon key="score">0.99939036</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:59:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11034" length="2"/>
<text>αB</text>
</annotation>
<annotation id="312">
<infon key="score">0.9995828</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11042" length="6"/>
<text>Arg521</text>
</annotation>
<annotation id="313">
<infon key="score">0.9992975</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11050" length="2"/>
<text>β5</text>
</annotation>
<annotation id="551">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:48Z</infon>
<location offset="11061" length="9"/>
<text>sub-patch</text>
</annotation>
<annotation id="314">
<infon key="score">0.9995974</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11085" length="6"/>
<text>His486</text>
</annotation>
<annotation id="315">
<infon key="score">0.9994097</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11093" length="2"/>
<text>αA</text>
</annotation>
<annotation id="316">
<infon key="score">0.9995894</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11098" length="6"/>
<text>Thr478</text>
</annotation>
<annotation id="317">
<infon key="score">0.9993623</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11106" length="2"/>
<text>β3</text>
</annotation>
<annotation id="318">
<infon key="score">0.99960214</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11111" length="6"/>
<text>Val524</text>
</annotation>
<annotation id="319">
<infon key="score">0.99926275</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11119" length="2"/>
<text>β5</text>
</annotation>
<annotation id="320">
<infon key="score">0.9995943</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11127" length="6"/>
<text>Phe534</text>
</annotation>
<annotation id="321">
<infon key="score">0.9993175</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11135" length="2"/>
<text>β6</text>
</annotation>
<annotation id="552">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:48Z</infon>
<location offset="11152" length="9"/>
<text>sub-patch</text>
</annotation>
<annotation id="322">
<infon key="score">0.8255207</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11176" length="19"/>
<text>first surface patch</text>
</annotation>
<annotation id="323">
<infon key="score">0.8227496</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11199" length="15"/>
<text>electropositive</text>
</annotation>
<annotation id="324">
<infon key="score">0.9995865</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11248" length="6"/>
<text>Arg477</text>
</annotation>
<annotation id="325">
<infon key="score">0.99958664</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:14:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11259" length="6"/>
<text>Tyr515</text>
</annotation>
<annotation id="326">
<infon key="score">0.99863356</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:16:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11275" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="327">
<infon key="score">0.9354888</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11292" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="507">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="11298" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="328">
<infon key="score">0.9981578</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11340" length="14"/>
<text>phosphorylated</text>
</annotation>
<annotation id="539">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:03:59Z</infon>
<location offset="11371" length="4"/>
<text>SMRT</text>
</annotation>
<annotation id="548">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:06:45Z</infon>
<location offset="11376" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="329">
<infon key="score">0.997118</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11404" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="330">
<infon key="score">0.9421021</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11448" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="508">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="11452" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="331">
<infon key="score">0.9987723</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:15:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11491" length="14"/>
<text>phosphorylated</text>
</annotation>
<annotation id="332">
<infon key="score">0.867387</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:16:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11539" length="20"/>
<text>second surface patch</text>
</annotation>
<annotation id="333">
<infon key="score">0.5920983</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:16:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11568" length="11"/>
<text>hydrophobic</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g004.jpg</infon>
<infon key="id">pone.0160694.g004</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>11638</offset>
<text>A conserved surface patch of FPA SPOC domain.</text>
<annotation id="334">
<infon key="score">0.92189</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:16:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11640" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="335">
<infon key="score">0.9779961</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11650" length="13"/>
<text>surface patch</text>
</annotation>
<annotation id="336">
<infon key="score">0.70229733</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11667" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="509">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="11671" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g004.jpg</infon>
<infon key="id">pone.0160694.g004</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>11684</offset>
<text>(A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</text>
<annotation id="337">
<infon key="score">0.99155676</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11727" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="338">
<infon key="score">0.9992993</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11731" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="339">
<infon key="score">0.99874276</infon>
<infon key="type">taxonomy_domain</infon>
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<infon key="updated_at">2023-09-20T12:49:28Z</infon>
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<passage>
<infon key="section_type">RESULTS</infon>
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<text>We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).</text>
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<text>Arg477</text>
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<infon key="updated_at">2023-09-20T13:14:15Z</infon>
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<infon key="updated_at">2023-09-20T12:52:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12398" length="13"/>
<text>surface patch</text>
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<infon key="updated_at">2023-09-20T13:17:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12428" length="9"/>
<text>conserved</text>
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<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
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<text>SPOC</text>
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<infon key="identifier">MESH:</infon>
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<text>introduced</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
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<text>FPA</text>
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<infon key="annotator">cleaner0</infon>
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<infon key="identifier">MESH:</infon>
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<text>stably transformed</text>
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<infon key="identifier">GENE:</infon>
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<text>fpa-8</text>
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<infon key="updated_at">2023-09-20T13:21:19Z</infon>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12778" length="3"/>
<text>FPA</text>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:20:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12845" length="21"/>
<text>expression constructs</text>
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<annotation id="365">
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<infon key="type">gene</infon>
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<infon key="updated_at">2023-09-20T13:18:58Z</infon>
<infon key="identifier">GENE:</infon>
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<text>fpa-8</text>
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<annotation id="366">
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:40Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>wild-type</text>
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<annotation id="367">
<infon key="score">0.9986777</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12908" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="368">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
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<text>FPA</text>
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<annotation id="556">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:21:09Z</infon>
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<text>expression levels</text>
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<infon key="updated_at">2023-09-20T13:19:39Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>wild-type</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13032" length="3"/>
<text>FPA</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:05:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13051" length="3"/>
<text>RNA</text>
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<infon key="score">0.9891585</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13088" length="3"/>
<text>FPA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:30Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>pre-mRNA</text>
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<infon key="updated_at">2023-09-20T13:20:34Z</infon>
<infon key="identifier">MESH:</infon>
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<text>R477A</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:20:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13189" length="5"/>
<text>Y515A</text>
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<annotation id="376">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:20:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13195" length="8"/>
<text>mutation</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13258" length="3"/>
<text>FPA</text>
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<annotation id="378">
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:40Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>wild-type</text>
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<infon key="updated_at">2023-09-20T13:20:50Z</infon>
<infon key="identifier">MESH:</infon>
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<text>protein blot</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
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<text>FPA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13409</offset>
<text>We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.</text>
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<text>surface patch</text>
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<infon key="updated_at">2023-09-20T15:07:57Z</infon>
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<text>mutations</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
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<text>FPA</text>
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<infon key="updated_at">2023-09-20T13:21:19Z</infon>
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<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<infon key="identifier">GENE:</infon>
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<text>FLC</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
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<text>FPA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13738" length="8"/>
<text>pre-mRNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:29:03Z</infon>
<infon key="identifier">MESH:</infon>
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<text>RNA gel blot analyses</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:21:19Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>mutant</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
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<text>FPA</text>
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<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>14166</offset>
<text>Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.</text>
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<infon key="type">fig_caption</infon>
<offset>14261</offset>
<text>RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A
(A), or FPA::FPA Y515A
(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A
(C), FPA::FPA Y515A
(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text>
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<text>qRT-PCR</text>
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<infon key="identifier">MESH:</infon>
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<text>PCR</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15153</offset>
<text>We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
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<offset>15573</offset>
<text>Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).</text>
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<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15777" length="3"/>
<text>FPA</text>
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<annotation id="560">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:32:13Z</infon>
<location offset="15859" length="15"/>
<text>FPA R477A;Y515A</text>
</annotation>
<annotation id="443">
<infon key="score">0.98018205</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:07:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15875" length="14"/>
<text>doubly mutated</text>
</annotation>
<annotation id="444">
<infon key="score">0.6438746</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:18:58Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="15904" length="5"/>
<text>fpa-8</text>
</annotation>
<annotation id="445">
<infon key="score">0.9978872</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:21:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15910" length="6"/>
<text>mutant</text>
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<annotation id="561">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:32:13Z</infon>
<location offset="15952" length="15"/>
<text>FPA R477A;Y515A</text>
</annotation>
<annotation id="446">
<infon key="score">0.99899787</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15992" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="447">
<infon key="score">0.9988796</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16002" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="448">
<infon key="score">0.9158664</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16017" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="449">
<infon key="score">0.9955418</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16021" length="8"/>
<text>pre-mRNA</text>
</annotation>
<annotation id="529">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<location offset="16068" length="3"/>
<text>FLC</text>
</annotation>
<annotation id="450">
<infon key="score">0.9989771</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16086" length="9"/>
<text>wild-type</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g006.jpg</infon>
<infon key="id">pone.0160694.g006</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>16113</offset>
<text>Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.</text>
<annotation id="451">
<infon key="score">0.8876505</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16130" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="514">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="16134" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="452">
<infon key="score">0.4253289</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16146" length="9"/>
<text>mutations</text>
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<annotation id="453">
<infon key="score">0.9432148</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16190" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="454">
<infon key="score">0.9954029</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:19:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16194" length="8"/>
<text>pre-mRNA</text>
</annotation>
<annotation id="455">
<infon key="score">0.6315412</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:56:39Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="16207" length="3"/>
<text>FLC</text>
</annotation>
</passage>
<passage>
<infon key="file">pone.0160694.g006.jpg</infon>
<infon key="id">pone.0160694.g006</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16223</offset>
<text>(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text>
<annotation id="456">
<infon key="score">0.9989862</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16227" length="12"/>
<text>RNA gel blot</text>
</annotation>
<annotation id="457">
<infon key="score">0.98714185</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:31:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16252" length="2"/>
<text>WT</text>
</annotation>
<annotation id="458">
<infon key="score">0.9781117</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:55:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16255" length="11"/>
<text>A. thaliana</text>
</annotation>
<annotation id="459">
<infon key="score">0.5267927</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:51:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16294" length="6"/>
<text>plants</text>
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<annotation id="460">
<infon key="score">0.7329895</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:18:58Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="16301" length="5"/>
<text>fpa-8</text>
</annotation>
<annotation id="461">
<infon key="score">0.8120683</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:18:58Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="16311" length="5"/>
<text>fpa-8</text>
</annotation>
<annotation id="462">
<infon key="score">0.9203812</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:30:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16317" length="7"/>
<text>mutants</text>
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<infon key="score">0.9772659</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16336" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="562">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:32:13Z</infon>
<location offset="16341" length="15"/>
<text>FPA R477A;Y515A</text>
</annotation>
<annotation id="464">
<infon key="score">0.99847776</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:53:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16381" length="5"/>
<text>mRNAs</text>
</annotation>
<annotation id="465">
<infon key="score">0.95337856</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16453" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="466">
<infon key="score">0.9983594</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:53:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16457" length="5"/>
<text>mRNAs</text>
</annotation>
<annotation id="467">
<infon key="score">0.94853</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16509" length="3"/>
<text>FPA</text>
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<annotation id="468">
<infon key="score">0.99780947</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:31:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16513" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="469">
<infon key="score">0.9346011</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16537" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="470">
<infon key="score">0.99581194</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:53:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16550" length="5"/>
<text>mRNAs</text>
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<infon key="score">0.99899334</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:30:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16714" length="7"/>
<text>qRT-PCR</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:34:56Z</infon>
<location offset="16756" length="3"/>
<text>RNA</text>
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<annotation id="555">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:18:58Z</infon>
<location offset="16781" length="5"/>
<text>fpa-8</text>
</annotation>
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<infon key="score">0.9812045</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16792" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="473">
<infon key="score">0.94803566</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:32:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16797" length="15"/>
<text>FPA R477A;Y515A</text>
</annotation>
<annotation id="474">
<infon key="score">0.9977831</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:51:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16813" length="6"/>
<text>plants</text>
</annotation>
<annotation id="565">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:35:12Z</infon>
<location offset="16875" length="10"/>
<text>Histograms</text>
</annotation>
<annotation id="475">
<infon key="score">0.9770333</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16929" length="3"/>
<text>PCR</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>16983</offset>
<text>Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.</text>
<annotation id="476">
<infon key="score">0.9994332</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17029" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="477">
<infon key="score">0.91164637</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17073" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="478">
<infon key="score">0.97791255</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:05:29Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17091" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="479">
<infon key="score">0.50058526</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17141" length="9"/>
<text>mutations</text>
</annotation>
<annotation id="480">
<infon key="score">0.60978085</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17211" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="515">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="17215" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="481">
<infon key="score">0.69148296</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:08:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17252" length="9"/>
<text>mutations</text>
</annotation>
<annotation id="482">
<infon key="score">0.99684554</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:52:20Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17269" length="5"/>
<text>SHARP</text>
</annotation>
<annotation id="483">
<infon key="score">0.99948525</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17275" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="484">
<infon key="score">0.99606663</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T15:07:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17317" length="16"/>
<text>unphosphorylated</text>
</annotation>
<annotation id="540">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T13:03:59Z</infon>
<location offset="17334" length="4"/>
<text>SMRT</text>
</annotation>
<annotation id="566">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T14:35:45Z</infon>
<location offset="17339" length="8"/>
<text>peptides</text>
</annotation>
<annotation id="485">
<infon key="score">0.79536206</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:29Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17430" length="3"/>
<text>FPA</text>
</annotation>
<annotation id="516">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="17434" length="4"/>
<text>SPOC</text>
</annotation>
<annotation id="517">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:49:04Z</infon>
<location offset="17501" length="4"/>
<text>SPOC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>17516</offset>
<text>Materials and Methods</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>17538</offset>
<text>Protein expression and purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>17574</offset>
<text>The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>18213</offset>
<text>The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>18376</offset>
<text>Protein crystallization</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>18400</offset>
<text>Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>18971</offset>
<text>Data collection and processing</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>19002</offset>
<text>A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>19402</offset>
<text>Structure determination and refinement</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>19441</offset>
<text>The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>20007</offset>
<text>Generation of constructs with mutated genomic FPA sequence</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>20066</offset>
<text>A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>20860</offset>
<text>Generation of Arabidopsis thaliana transgenic plants</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>20913</offset>
<text>All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>21390</offset>
<text>Plant growth conditions</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>21414</offset>
<text>Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>22514</offset>
<text>Plant protein analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>22537</offset>
<text>Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>24024</offset>
<text>RNA gel blot analysis and RT-qPCR</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>24058</offset>
<text>RNA gel blot analysis and RT-qPCR method performed as previously described.</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>24134</offset>
<text>Supporting Information</text>
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<passage>
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<infon key="type">title</infon>
<offset>24157</offset>
<text>References</text>
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<infon key="article-id_doi">10.1038/srep31425</infon>
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<infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon>
<infon key="name_0">surname:Chen;given-names:Ji-Yun</infon>
<infon key="name_1">surname:Liu;given-names:Liang</infon>
<infon key="name_2">surname:Cao;given-names:Chun-Ling</infon>
<infon key="name_3">surname:Li;given-names:Mei-Jun</infon>
<infon key="name_4">surname:Tan;given-names:Kemin</infon>
<infon key="name_5">surname:Yang;given-names:Xiaohan</infon>
<infon key="name_6">surname:Yun;given-names:Cai-Hong</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">6</infon>
<infon key="year">2016</infon>
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<text>Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase</text>
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<text>human</text>
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<infon key="updated_at">2023-09-20T09:44:46Z</infon>
<infon key="identifier">PR:</infon>
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<text>Naa60</text>
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<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="39" length="4"/>
<text>NatF</text>
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<infon key="score">0.9983005</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="78" length="17"/>
<text>acetyltransferase</text>
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<passage>
<infon key="section_type">ABSTRACT</infon>
<infon key="type">abstract</infon>
<offset>96</offset>
<text>N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.</text>
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<infon key="score">0.9378259</infon>
<infon key="type">ptm</infon>
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<infon key="updated_at">2023-09-20T09:45:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="96" length="22"/>
<text>N-terminal acetylation</text>
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<infon key="score">0.9623558</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="120" length="14"/>
<text>Nt-acetylation</text>
</annotation>
<annotation id="7">
<infon key="score">0.99854547</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="152" length="29"/>
<text>N-terminal acetyltransferases</text>
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<annotation id="8">
<infon key="score">0.99888486</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="183" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="985">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="241" length="7"/>
<text>peptide</text>
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<infon key="score">0.9994029</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="257" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="10">
<infon key="score">0.99875224</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="275" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="11">
<infon key="score">0.99907637</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="306" length="3"/>
<text>NAT</text>
</annotation>
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<infon key="score">0.97256976</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="324" length="24"/>
<text>multicellular eukaryotes</text>
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<infon key="score">0.96585155</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="430" length="14"/>
<text>Nt-acetylation</text>
</annotation>
<annotation id="14">
<infon key="score">0.9983275</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="492" length="27"/>
<text>lysine Nε-acetyltransferase</text>
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<annotation id="15">
<infon key="score">0.99864644</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="521" length="3"/>
<text>KAT</text>
</annotation>
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<infon key="score">0.99024945</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="551" length="11"/>
<text>acetylation</text>
</annotation>
<annotation id="17">
<infon key="score">0.99478674</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="566" length="6"/>
<text>lysine</text>
</annotation>
<annotation id="18">
<infon key="score">0.9975256</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="602" length="18"/>
<text>crystal structures</text>
</annotation>
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<infon key="score">0.99812895</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="624" length="5"/>
<text>human</text>
</annotation>
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<infon key="score">0.9994475</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="630" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="21">
<infon key="score">0.99933773</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="637" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="22">
<infon key="score">0.99842304</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="645" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="23">
<infon key="score">0.99915624</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="661" length="17"/>
<text>Acetyl-Coenzyme A</text>
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<infon key="updated_at">2023-09-20T12:29:42Z</infon>
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<location offset="772" length="11"/>
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<text>1-242</text>
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<location offset="994" length="6"/>
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<location offset="1222" length="7"/>
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<text>Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.</text>
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<text>Acetylation</text>
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<location offset="1657" length="11"/>
<text>acetylation</text>
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<annotation id="894">
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<text>lysine</text>
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<infon key="updated_at">2023-09-20T09:49:17Z</infon>
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<infon key="updated_at">2023-09-20T09:49:22Z</infon>
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<location offset="1999" length="26"/>
<text>histone acetyltransferases</text>
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<infon key="updated_at">2023-09-20T09:49:33Z</infon>
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<location offset="2027" length="4"/>
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<infon key="updated_at">2023-09-20T09:48:10Z</infon>
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<location offset="2098" length="11"/>
<text>acetylation</text>
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<annotation id="53">
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<infon key="updated_at">2023-09-20T09:49:40Z</infon>
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<location offset="2113" length="8"/>
<text>histones</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2141</offset>
<text>Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.</text>
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<infon key="updated_at">2023-09-20T09:48:54Z</infon>
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<location offset="2202" length="14"/>
<text>Nε-acetylation</text>
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<infon key="updated_at">2023-09-20T09:49:23Z</infon>
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<location offset="2220" length="4"/>
<text>KATs</text>
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<infon key="updated_at">2023-09-20T09:45:10Z</infon>
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<location offset="2298" length="14"/>
<text>Nt-acetylation</text>
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<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:10Z</infon>
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<location offset="2336" length="14"/>
<text>Nt-acetylation</text>
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<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:24:05Z</infon>
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<location offset="2421" length="8"/>
<text>bacteria</text>
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<infon key="updated_at">2023-09-20T12:24:08Z</infon>
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<location offset="2431" length="7"/>
<text>archaea</text>
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<annotation id="60">
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<infon key="updated_at">2023-09-20T12:24:11Z</infon>
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<location offset="2443" length="10"/>
<text>eukaryotes</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:22Z</infon>
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<location offset="2500" length="5"/>
<text>human</text>
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<infon key="updated_at">2023-09-20T09:50:01Z</infon>
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<location offset="2529" length="5"/>
<text>yeast</text>
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<infon key="updated_at">2023-09-20T09:45:10Z</infon>
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<location offset="2753" length="14"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:50:49Z</infon>
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<location offset="2794" length="12"/>
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<infon key="updated_at">2023-09-20T12:26:48Z</infon>
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<location offset="3037" length="14"/>
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<infon key="updated_at">2023-09-20T09:45:10Z</infon>
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<location offset="3170" length="14"/>
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<infon key="updated_at">2023-09-20T09:51:24Z</infon>
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<text>plant</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3376</offset>
<text>The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.</text>
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<text>Nt-acetylation</text>
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<text>C</text>
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<text>D</text>
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<text>E</text>
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<text>F</text>
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<text>eukaryotes</text>
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<text>Nt-acetylation</text>
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<text>NatA</text>
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<text>NatE</text>
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<location offset="3782" length="4"/>
<text>NatA</text>
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<text>NatA</text>
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<text>NATs</text>
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<text>NatB</text>
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<infon key="updated_at">2023-09-20T09:54:58Z</infon>
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<text>NatC</text>
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<text>Naa30</text>
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<infon key="updated_at">2023-09-20T09:53:14Z</infon>
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<location offset="3984" length="5"/>
<text>Naa25</text>
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<infon key="updated_at">2023-09-20T09:53:19Z</infon>
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<location offset="3994" length="5"/>
<text>Naa35</text>
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<infon key="score">0.99870276</infon>
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<infon key="updated_at">2023-09-20T09:53:23Z</infon>
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<location offset="4000" length="5"/>
<text>Naa38</text>
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<infon key="score">0.9986804</infon>
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<infon key="updated_at">2023-09-20T09:53:28Z</infon>
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<text>Naa60</text>
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<infon key="updated_at">2023-09-20T09:55:17Z</infon>
<infon key="identifier">GO:</infon>
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<text>NatD</text>
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<annotation id="104">
<infon key="score">0.9985499</infon>
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<infon key="updated_at">2023-09-20T09:55:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="4102" length="4"/>
<text>NatF</text>
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<annotation id="105">
<infon key="score">0.96562463</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4130" length="14"/>
<text>Nt-acetylation</text>
</annotation>
<annotation id="106">
<infon key="score">0.9625132</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4181" length="14"/>
<text>Nε-acetylation</text>
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<annotation id="107">
<infon key="score">0.99909365</infon>
<infon key="type">protein_type</infon>
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<infon key="updated_at">2023-09-20T09:45:22Z</infon>
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<text>NATs</text>
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</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>4223</offset>
<text>There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.</text>
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<text>Nt-acetylation</text>
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<infon key="score">0.9985532</infon>
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<infon key="updated_at">2023-09-20T09:50:02Z</infon>
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<text>yeast</text>
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<text>human</text>
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<infon key="updated_at">2023-09-20T09:55:36Z</infon>
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<location offset="4370" length="4"/>
<text>NatF</text>
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<infon key="score">0.99850816</infon>
<infon key="type">protein_type</infon>
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<infon key="updated_at">2023-09-20T10:02:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4389" length="28"/>
<text>N-terminal acetyltransferase</text>
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<infon key="score">0.99880755</infon>
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<infon key="updated_at">2023-09-20T09:55:36Z</infon>
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<text>NatF</text>
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<text>NAA60</text>
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<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-20T10:03:06Z</infon>
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<text>Histone acetyltransferase type B protein 4</text>
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<infon key="updated_at">2023-09-20T10:03:53Z</infon>
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<text>N-acetyltransferase 15</text>
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<infon key="score">0.9983645</infon>
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<infon key="updated_at">2023-09-20T10:03:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4571" length="5"/>
<text>NAT15</text>
</annotation>
<annotation id="897">
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<text>NAT</text>
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<infon key="updated_at">2023-09-20T09:45:22Z</infon>
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<location offset="4634" length="4"/>
<text>NATs</text>
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<infon key="updated_at">2023-09-20T12:26:56Z</infon>
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<text>highly conserved</text>
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<infon key="updated_at">2023-09-20T12:24:19Z</infon>
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<text>lower</text>
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<annotation id="121">
<infon key="score">0.8346692</infon>
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<infon key="updated_at">2023-09-20T10:06:05Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>higher eukaryotes</text>
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<infon key="updated_at">2023-09-20T09:55:36Z</infon>
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<location offset="4700" length="4"/>
<text>NatF</text>
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<infon key="score">0.98239946</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:06:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4720" length="17"/>
<text>higher eukaryotes</text>
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<infon key="score">0.99875</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="4776" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="125">
<infon key="score">0.9238295</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4822" length="14"/>
<text>Nt-acetylation</text>
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<annotation id="919">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:07:04Z</infon>
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<text>lysine Nε-acetylation</text>
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<annotation id="126">
<infon key="score">0.9984901</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:02:42Z</infon>
<infon key="identifier">MESH:</infon>
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<text>N-terminal acetyltransferase</text>
</annotation>
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<infon key="score">0.99684405</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="4900" length="4"/>
<text>NatF</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4931" length="11"/>
<text>acetylation</text>
</annotation>
<annotation id="933">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:14:47Z</infon>
<location offset="5051" length="8"/>
<text>Met-Lys-</text>
</annotation>
<annotation id="934">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:15:08Z</infon>
<location offset="5061" length="8"/>
<text>Met-Val-</text>
</annotation>
<annotation id="935">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:15:27Z</infon>
<location offset="5071" length="8"/>
<text>Met-Ala-</text>
</annotation>
<annotation id="936">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:15:45Z</infon>
<location offset="5084" length="8"/>
<text>Met-Met-</text>
</annotation>
<annotation id="129">
<infon key="score">0.9971757</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:54:58Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5157" length="4"/>
<text>NatC</text>
</annotation>
<annotation id="130">
<infon key="score">0.99452823</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:00:06Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5166" length="4"/>
<text>NatE</text>
</annotation>
<annotation id="131">
<infon key="score">0.9983417</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5191" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="132">
<infon key="score">0.99512225</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:08:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5206" length="24"/>
<text>lysine acetyltransferase</text>
</annotation>
<annotation id="917">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:05:36Z</infon>
<location offset="5254" length="18"/>
<text>lysine acetylation</text>
</annotation>
<annotation id="133">
<infon key="score">0.98459953</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:08:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5281" length="7"/>
<text>histone</text>
</annotation>
<annotation id="134">
<infon key="score">0.9713598</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:09:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5289" length="2"/>
<text>H4</text>
</annotation>
<annotation id="926">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:11:31Z</infon>
<location offset="5303" length="2"/>
<text>H4</text>
</annotation>
<annotation id="927">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:11:44Z</infon>
<location offset="5305" length="3"/>
<text>K20</text>
</annotation>
<annotation id="928">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:12:04Z</infon>
<location offset="5310" length="2"/>
<text>H4</text>
</annotation>
<annotation id="929">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:12:28Z</infon>
<location offset="5312" length="3"/>
<text>K79</text>
</annotation>
<annotation id="930">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:12:49Z</infon>
<location offset="5320" length="2"/>
<text>H4</text>
</annotation>
<annotation id="931">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:05Z</infon>
<location offset="5322" length="3"/>
<text>K91</text>
</annotation>
<annotation id="135">
<infon key="score">0.9989195</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5356" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="136">
<infon key="score">0.9942046</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:29:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5440" length="27"/>
<text>membrane-integrating region</text>
</annotation>
<annotation id="137">
<infon key="score">0.9988495</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5603" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="898">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<location offset="5667" length="3"/>
<text>NAT</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>5697</offset>
<text>It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.</text>
<annotation id="138">
<infon key="score">0.939754</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5724" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="139">
<infon key="score">0.9885904</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:22:55Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5741" length="11"/>
<text>nucleosomes</text>
</annotation>
<annotation id="140">
<infon key="score">0.89290154</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5771" length="5"/>
<text>NAA60</text>
</annotation>
<annotation id="141">
<infon key="score">0.9981092</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5891" length="10"/>
<text>Drosophila</text>
</annotation>
<annotation id="142">
<infon key="score">0.7164299</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5909" length="5"/>
<text>NAA60</text>
</annotation>
<annotation id="143">
<infon key="score">0.9589997</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="6102" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="144">
<infon key="score">0.79295474</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6162" length="5"/>
<text>NAA60</text>
</annotation>
<annotation id="145">
<infon key="score">0.9425036</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6239" length="14"/>
<text>overexpression</text>
</annotation>
<annotation id="146">
<infon key="score">0.99772257</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6254" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="147">
<infon key="score">0.9991636</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6340" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="148">
<infon key="score">0.99801564</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6399" length="10"/>
<text>Drosophila</text>
</annotation>
<annotation id="149">
<infon key="score">0.9858074</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="6414" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="150">
<infon key="score">0.99814355</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6480" length="10"/>
<text>Drosophila</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>6492</offset>
<text>In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.</text>
<annotation id="151">
<infon key="score">0.9637387</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6510" length="6"/>
<text>solved</text>
</annotation>
<annotation id="152">
<infon key="score">0.94823563</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6521" length="10"/>
<text>structures</text>
</annotation>
<annotation id="153">
<infon key="score">0.9984149</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6535" length="5"/>
<text>human</text>
</annotation>
<annotation id="154">
<infon key="score">0.9993923</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6541" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="155">
<infon key="score">0.99935895</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="6548" length="4"/>
<text>NatF</text>
</annotation>
<annotation id="156">
<infon key="score">0.9983024</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6554" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="157">
<infon key="score">0.9977319</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6570" length="8"/>
<text>coenzyme</text>
</annotation>
<annotation id="158">
<infon key="score">0.9993267</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6584" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="1038">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:27:15Z</infon>
<location offset="6617" length="19"/>
<text>amphipathic α-helix</text>
</annotation>
<annotation id="159">
<infon key="score">0.9994553</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:29:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6638" length="2"/>
<text>α5</text>
</annotation>
<annotation id="160">
<infon key="score">0.99896514</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:29:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6656" length="11"/>
<text>GNAT domain</text>
</annotation>
<annotation id="161">
<infon key="score">0.99477154</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6731" length="18"/>
<text>Crystal structures</text>
</annotation>
<annotation id="162">
<infon key="score">0.99920386</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6766" length="13"/>
<text>β7-β8 hairpin</text>
</annotation>
<annotation id="1048">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:36:24Z</infon>
<location offset="6823" length="17"/>
<text>C-terminal region</text>
</annotation>
<annotation id="163">
<infon key="score">0.99904025</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6916" length="24"/>
<text>substrate-binding pocket</text>
</annotation>
<annotation id="164">
<infon key="score">0.9975388</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6967" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="165">
<infon key="score">0.87373805</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:13:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7023" length="8"/>
<text>coenzyme</text>
</annotation>
<annotation id="166">
<infon key="score">0.99868906</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7076" length="20"/>
<text>structure comparison</text>
</annotation>
<annotation id="167">
<infon key="score">0.9906101</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:15:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7101" length="19"/>
<text>biochemical studies</text>
</annotation>
<annotation id="168">
<infon key="score">0.99938595</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7204" length="5"/>
<text>Naa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>7225</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>7233</offset>
<text>Overall structure of hNaa60</text>
<annotation id="169">
<infon key="score">0.9977296</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:16:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7241" length="9"/>
<text>structure</text>
</annotation>
<annotation id="170">
<infon key="score">0.99925464</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7254" length="6"/>
<text>hNaa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>7261</offset>
<text>In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).</text>
<annotation id="171">
<infon key="score">0.99925226</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7349" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="172">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7425" length="18"/>
<text>Sequence alignment</text>
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<annotation id="173">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
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<location offset="7447" length="5"/>
<text>Naa60</text>
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<annotation id="939">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:18:40Z</infon>
<location offset="7487" length="11"/>
<text>Glu-Glu-Arg</text>
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<annotation id="174">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:18:49Z</infon>
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<location offset="7500" length="3"/>
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<annotation id="940">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:16Z</infon>
<location offset="7512" length="11"/>
<text>Val-Val-Pro</text>
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<annotation id="175">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7525" length="3"/>
<text>VVP</text>
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<annotation id="176">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7588" length="14"/>
<text>Xenopus Laevis</text>
</annotation>
<annotation id="177">
<infon key="score">0.9984832</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7610" length="12"/>
<text>Homo sapiens</text>
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<annotation id="178">
<infon key="score">0.9906036</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7803" length="7"/>
<text>mutated</text>
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<annotation id="179">
<infon key="score">0.9951412</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:32:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7820" length="3"/>
<text>4–6</text>
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<annotation id="938">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:18:21Z</infon>
<location offset="7829" length="10"/>
<text>VVP to EER</text>
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<annotation id="180">
<infon key="score">0.9993231</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7977" length="6"/>
<text>hNaa60</text>
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<annotation id="181">
<infon key="score">0.99922884</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8020" length="6"/>
<text>hNaa60</text>
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<annotation id="182">
<infon key="score">0.8594266</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8062" length="8"/>
<text>mutation</text>
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<annotation id="183">
<infon key="score">0.99651885</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8084" length="9"/>
<text>truncated</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:33Z</infon>
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<location offset="8102" length="5"/>
<text>1-199</text>
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<infon key="score">0.99910736</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8116" length="11"/>
<text>full-length</text>
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<annotation id="186">
<infon key="score">0.9986326</infon>
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<infon key="updated_at">2023-09-20T12:25:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8166" length="7"/>
<text>crystal</text>
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<infon key="score">0.9977609</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8181" length="9"/>
<text>truncated</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:54Z</infon>
<location offset="8199" length="5"/>
<text>1-199</text>
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<annotation id="188">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8205" length="15"/>
<text>in complex with</text>
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<annotation id="189">
<infon key="score">0.998934</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8221" length="3"/>
<text>CoA</text>
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<annotation id="190">
<infon key="score">0.99869686</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8270" length="7"/>
<text>crystal</text>
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<infon key="score">0.99911183</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8285" length="11"/>
<text>full-length</text>
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<annotation id="192">
<infon key="score">0.9829355</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8324" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="193">
<infon key="score">0.9957215</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8331" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="194">
<infon key="score">0.9988327</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="8347" length="6"/>
<text>Ac-CoA</text>
</annotation>
<annotation id="1010">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<location offset="8401" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="195">
<infon key="score">0.99930966</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8412" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="196">
<infon key="score">0.6943366</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8449" length="3"/>
<text>EER</text>
</annotation>
<annotation id="942">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:07Z</infon>
<location offset="8453" length="8"/>
<text>mutation</text>
</annotation>
<annotation id="197">
<infon key="score">0.99876124</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8467" length="18"/>
<text>crystal structures</text>
</annotation>
<annotation id="944">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:34Z</infon>
<location offset="8489" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="951">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:53Z</infon>
<location offset="8514" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="198">
<infon key="score">0.9986177</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:17:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8551" length="21"/>
<text>molecular replacement</text>
</annotation>
<annotation id="199">
<infon key="score">0.9987355</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8646" length="21"/>
<text>electron density maps</text>
</annotation>
<annotation id="200">
<infon key="score">0.9942498</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:32:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8713" length="5"/>
<text>1-211</text>
</annotation>
<annotation id="201">
<infon key="score">0.8964569</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8722" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="937">
<infon key="type">residue_range</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:16:33Z</infon>
<location offset="8729" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="202">
<infon key="score">0.99425864</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:32:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8749" length="5"/>
<text>5-199</text>
</annotation>
<annotation id="1043">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:32:35Z</infon>
<location offset="8758" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>8783</offset>
<text>The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).</text>
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<infon key="score">0.9957825</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8787" length="9"/>
<text>structure</text>
</annotation>
<annotation id="204">
<infon key="score">0.9993191</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8800" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="205">
<infon key="score">0.99772197</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8826" length="14"/>
<text>central domain</text>
</annotation>
<annotation id="206">
<infon key="score">0.9719784</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8862" length="32"/>
<text>GCN5-related N-acetyltransferase</text>
</annotation>
<annotation id="207">
<infon key="score">0.9992061</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8896" length="4"/>
<text>GNAT</text>
</annotation>
<annotation id="208">
<infon key="score">0.7512091</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8926" length="8"/>
<text>extended</text>
</annotation>
<annotation id="209">
<infon key="score">0.9954179</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8935" length="25"/>
<text>N- and C-terminal regions</text>
</annotation>
<annotation id="210">
<infon key="score">0.9985126</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8978" length="14"/>
<text>central domain</text>
</annotation>
<annotation id="211">
<infon key="score">0.9941509</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9007" length="9"/>
<text>β strands</text>
</annotation>
<annotation id="212">
<infon key="score">0.9976589</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9018" length="5"/>
<text>β1-β9</text>
</annotation>
<annotation id="213">
<infon key="score">0.98797673</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9034" length="9"/>
<text>α-helixes</text>
</annotation>
<annotation id="214">
<infon key="score">0.99689</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9045" length="5"/>
<text>α1-α4</text>
</annotation>
<annotation id="215">
<infon key="score">0.99236286</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9059" length="14"/>
<text>highly similar</text>
</annotation>
<annotation id="216">
<infon key="score">0.99932754</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9087" length="7"/>
<text>hNaa50p</text>
</annotation>
<annotation id="217">
<infon key="score">0.99929893</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9114" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="218">
<infon key="score">0.9993673</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9142" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="219">
<infon key="score">0.9921711</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9162" length="21"/>
<text>extra 20-residue loop</text>
</annotation>
<annotation id="220">
<infon key="score">0.999292</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9192" length="2"/>
<text>β3</text>
</annotation>
<annotation id="221">
<infon key="score">0.9991374</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9199" length="2"/>
<text>β4</text>
</annotation>
<annotation id="222">
<infon key="score">0.9988525</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9215" length="15"/>
<text>small subdomain</text>
</annotation>
<annotation id="223">
<infon key="score">0.99903136</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9292" length="13"/>
<text>β7-β8 strands</text>
</annotation>
<annotation id="224">
<infon key="score">0.9855807</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:22:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9314" length="46"/>
<text>approximately antiparallel β-hairpin structure</text>
</annotation>
<annotation id="225">
<infon key="score">0.99932003</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9395" length="7"/>
<text>hNaa50p</text>
</annotation>
<annotation id="226">
<infon key="score">0.9703472</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9418" length="25"/>
<text>N- and C-terminal regions</text>
</annotation>
<annotation id="227">
<infon key="score">0.8584138</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9449" length="18"/>
<text>helical structures</text>
</annotation>
<annotation id="228">
<infon key="score">0.99945825</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9469" length="2"/>
<text>α0</text>
</annotation>
<annotation id="229">
<infon key="score">0.9993788</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9476" length="2"/>
<text>α5</text>
</annotation>
<annotation id="230">
<infon key="score">0.99567384</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9512" length="11"/>
<text>GCN5-domain</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9579</offset>
<text>Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).</text>
<annotation id="231">
<infon key="score">0.9958061</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9634" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="232">
<infon key="score">0.993637</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9641" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="965">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:44Z</infon>
<location offset="9675" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="233">
<infon key="score">0.99601203</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:33:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9727" length="7"/>
<text>200–242</text>
</annotation>
<annotation id="903">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<location offset="9853" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="959">
<infon key="type">residue_range</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<location offset="9860" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="234">
<infon key="score">0.99869287</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9867" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="235">
<infon key="score">0.99932134</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9963" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="236">
<infon key="score">0.99910516</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10036" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="237">
<infon key="score">0.9993279</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10048" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="1011">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<location offset="10189" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="966">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:37Z</infon>
<location offset="10202" length="19"/>
<text>mutagenesis studies</text>
</annotation>
<annotation id="1044">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:33:03Z</infon>
<location offset="10260" length="14"/>
<text>hNaa60 (1-199)</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>10278</offset>
<text>An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60</text>
<annotation id="1039">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:27:40Z</infon>
<location offset="10281" length="19"/>
<text>amphipathic α-helix</text>
</annotation>
<annotation id="238">
<infon key="score">0.996947</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10308" length="17"/>
<text>C-terminal region</text>
</annotation>
<annotation id="239">
<infon key="score">0.9990615</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10366" length="6"/>
<text>hNaa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10377</offset>
<text>There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).</text>
<annotation id="240">
<infon key="score">0.9991192</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10390" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="241">
<infon key="score">0.9910477</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="10436" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="242">
<infon key="score">0.9924954</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10457" length="9"/>
<text>structure</text>
</annotation>
<annotation id="243">
<infon key="score">0.99688613</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10472" length="17"/>
<text>C-terminal region</text>
</annotation>
<annotation id="244">
<infon key="score">0.99803805</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:25:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10508" length="11"/>
<text>GCN5-domain</text>
</annotation>
<annotation id="245">
<infon key="score">0.9984503</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10529" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="246">
<infon key="score">0.99945563</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10548" length="2"/>
<text>α5</text>
</annotation>
<annotation id="247">
<infon key="score">0.9969907</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10620" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
<annotation id="248">
<infon key="score">0.9992375</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10653" length="8"/>
<text>α5-helix</text>
</annotation>
<annotation id="249">
<infon key="score">0.99887705</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10668" length="18"/>
<text>hydrophobic groove</text>
</annotation>
<annotation id="250">
<infon key="score">0.99939835</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10710" length="2"/>
<text>β1</text>
</annotation>
<annotation id="251">
<infon key="score">0.99835706</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10717" length="10"/>
<text>β3 strands</text>
</annotation>
<annotation id="252">
<infon key="score">0.9985872</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10768" length="20"/>
<text>C-terminal extension</text>
</annotation>
<annotation id="253">
<infon key="score">0.9992624</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10799" length="8"/>
<text>α5-helix</text>
</annotation>
<annotation id="254">
<infon key="score">0.99928707</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10816" length="6"/>
<text>β-turn</text>
</annotation>
<annotation id="255">
<infon key="score">0.9970007</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10898" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
<annotation id="256">
<infon key="score">0.9390323</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<infon key="identifier">GO:</infon>
<location offset="10936" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="257">
<infon key="score">0.99651355</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10954" length="9"/>
<text>structure</text>
</annotation>
<annotation id="258">
<infon key="score">0.99932104</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10979" length="8"/>
<text>α5-helix</text>
</annotation>
<annotation id="259">
<infon key="score">0.9868099</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11024" length="17"/>
<text>C-terminal region</text>
</annotation>
<annotation id="260">
<infon key="score">0.9992686</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11094" length="8"/>
<text>α5-helix</text>
</annotation>
<annotation id="968">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:48Z</infon>
<location offset="11117" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="261">
<infon key="score">0.9966192</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11148" length="23"/>
<text>hydrophobic interaction</text>
</annotation>
<annotation id="262">
<infon key="score">0.9988097</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11192" length="18"/>
<text>hydrophobic groove</text>
</annotation>
<annotation id="263">
<infon key="score">0.9987142</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11255" length="5"/>
<text>helix</text>
</annotation>
<annotation id="980">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:44:17Z</infon>
<location offset="11308" length="15"/>
<text>crystal packing</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>11364</offset>
<text>A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.</text>
<annotation id="264">
<infon key="score">0.99668574</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11403" length="7"/>
<text>182–216</text>
</annotation>
<annotation id="265">
<infon key="score">0.9992968</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11449" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="266">
<infon key="score">0.99788886</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11483" length="9"/>
<text>structure</text>
</annotation>
<annotation id="267">
<infon key="score">0.8807922</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11498" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="268">
<infon key="score">0.9991995</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11514" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="269">
<infon key="score">0.99952435</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11533" length="2"/>
<text>α5</text>
</annotation>
<annotation id="270">
<infon key="score">0.99654055</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11556" length="7"/>
<text>190-202</text>
</annotation>
<annotation id="271">
<infon key="score">0.99106693</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:30Z</infon>
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<text>Ile 190</text>
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<text>Ile 194</text>
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<infon key="updated_at">2023-09-20T10:28:44Z</infon>
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<text>Leu 197</text>
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<passage>
<infon key="section_type">RESULTS</infon>
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<text>The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures</text>
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<text>β7-β8 hairpin</text>
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<text>crystal structures</text>
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<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
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<text>Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).</text>
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<text>Superposition</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12196" length="13"/>
<text>β7-β8 hairpin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:50Z</infon>
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<location offset="12269" length="11"/>
<text>GNAT domain</text>
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<text>hNaa60</text>
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<infon key="annotator">cleaner0</infon>
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<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12314" length="13"/>
<text>β7-β8 hairpin</text>
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<annotation id="287">
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<infon key="updated_at">2023-09-20T10:30:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12365" length="10"/>
<text>α1-α2 loop</text>
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<annotation id="288">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12401" length="22"/>
<text>substrate binding site</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
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<text>hNaa50</text>
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<infon key="updated_at">2023-09-20T10:30:38Z</infon>
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<location offset="12477" length="8"/>
<text>flexible</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:39Z</infon>
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<location offset="12486" length="4"/>
<text>loop</text>
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<annotation id="292">
<infon key="score">0.99916404</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12505" length="10"/>
<text>β6-β7 loop</text>
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<annotation id="293">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12523" length="8"/>
<text>removing</text>
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<annotation id="1047">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:36:03Z</infon>
<location offset="12536" length="17"/>
<text>C-terminal region</text>
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<infon key="updated_at">2023-09-20T09:46:45Z</infon>
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<location offset="12557" length="6"/>
<text>hNaa60</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12582" length="14"/>
<text>hNaa60 (1-199)</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
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<location offset="12645" length="13"/>
<text>β7-β8 hairpin</text>
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<annotation id="297">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:57Z</infon>
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<location offset="12666" length="7"/>
<text>crystal</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:31:01Z</infon>
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<location offset="12718" length="7"/>
<text>hairpin</text>
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<infon key="score">0.9992585</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12752" length="10"/>
<text>α1-α2 loop</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12821</offset>
<text>This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.</text>
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<infon key="updated_at">2023-09-20T12:39:21Z</infon>
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<text>substrate binding site</text>
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<infon key="updated_at">2023-09-20T10:34:00Z</infon>
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<text>active site</text>
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<text>hNaa60</text>
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<infon key="updated_at">2023-09-20T09:47:46Z</infon>
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<text>β7-β8 hairpin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:59Z</infon>
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<infon key="updated_at">2023-09-20T12:20:55Z</infon>
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<infon key="updated_at">2023-09-20T10:34:00Z</infon>
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<infon key="updated_at">2023-09-20T10:34:22Z</infon>
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<infon key="updated_at">2023-09-20T09:47:04Z</infon>
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<infon key="updated_at">2023-09-20T09:47:15Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
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<text>NAT</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
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<text>hNaa60</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:08:43Z</infon>
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<text>histone</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:09:14Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:10Z</infon>
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<text>enzyme kinetic data</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
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<location offset="13465" length="6"/>
<text>hNaa60</text>
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<infon key="updated_at">2023-09-20T10:32:12Z</infon>
<infon key="identifier">GO:</infon>
<location offset="13486" length="5"/>
<text>H3-H4</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13492" length="8"/>
<text>tetramer</text>
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<annotation id="316">
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<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13552" length="11"/>
<text>acetylation</text>
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<passage>
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<text>Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15559</offset>
<text>The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).</text>
<annotation id="358">
<infon key="score">0.9986228</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15563" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="359">
<infon key="score">0.99366796</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15583" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="946">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<location offset="15624" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="360">
<infon key="score">0.99336183</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15645" length="9"/>
<text>structure</text>
</annotation>
<annotation id="953">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<location offset="15685" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="361">
<infon key="score">0.9961402</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15703" length="9"/>
<text>structure</text>
</annotation>
<annotation id="362">
<infon key="score">0.9921007</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15745" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="363">
<infon key="score">0.9992324</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15808" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="364">
<infon key="score">0.9924878</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15842" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="365">
<infon key="score">0.99913627</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15857" length="13"/>
<text>ethanethioate</text>
</annotation>
<annotation id="366">
<infon key="score">0.9987211</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15881" length="6"/>
<text>Ac-CoA</text>
</annotation>
<annotation id="367">
<infon key="score">0.90510833</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="15911" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="368">
<infon key="score">0.99532664</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15932" length="9"/>
<text>structure</text>
</annotation>
<annotation id="369">
<infon key="score">0.99804014</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15953" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="370">
<infon key="score">0.99360776</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15975" length="9"/>
<text>structure</text>
</annotation>
<annotation id="371">
<infon key="score">0.9954337</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:36:25Z</infon>
<infon key="identifier">GO:</infon>
<location offset="15996" length="19"/>
<text>hNaa50p/CoA/peptide</text>
</annotation>
<annotation id="372">
<infon key="score">0.99910945</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16031" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="373">
<infon key="score">0.9969879</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16081" length="10"/>
<text>methionine</text>
</annotation>
<annotation id="990">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="16109" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="374">
<infon key="score">0.99384165</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16129" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="375">
<infon key="score">0.9994134</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:45Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16139" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="376">
<infon key="score">0.9929946</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16163" length="6"/>
<text>Phe 27</text>
</annotation>
<annotation id="377">
<infon key="score">0.9994166</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16173" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="378">
<infon key="score">0.99719894</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16213" length="9"/>
<text>structure</text>
</annotation>
<annotation id="954">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<location offset="16226" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="379">
<infon key="score">0.9990219</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16267" length="3"/>
<text>CoA</text>
</annotation>
<annotation id="380">
<infon key="score">0.52795213</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:23:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16338" length="5"/>
<text>amine</text>
</annotation>
<annotation id="381">
<infon key="score">0.9921836</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:39:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16365" length="12"/>
<text>superimposed</text>
</annotation>
<annotation id="382">
<infon key="score">0.9987024</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:38:27Z</infon>
<infon key="identifier">GO:</infon>
<location offset="16378" length="18"/>
<text>hNaa50/CoA/peptide</text>
</annotation>
<annotation id="383">
<infon key="score">0.984336</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16397" length="9"/>
<text>structure</text>
</annotation>
<annotation id="384">
<infon key="score">0.9991757</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16433" length="13"/>
<text>ethanethioate</text>
</annotation>
<annotation id="385">
<infon key="score">0.9990859</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16450" length="6"/>
<text>Ac-CoA</text>
</annotation>
<annotation id="386">
<infon key="score">0.99643755</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16464" length="9"/>
<text>structure</text>
</annotation>
<annotation id="947">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<location offset="16477" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="387">
<infon key="score">0.9990897</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16528" length="10"/>
<text>α1-α2 loop</text>
</annotation>
<annotation id="388">
<infon key="score">0.9987115</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16621" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="389">
<infon key="score">0.99693334</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16714" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="390">
<infon key="score">0.99336606</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16762" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="391">
<infon key="score">0.9901281</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:36:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16773" length="6"/>
<text>solved</text>
</annotation>
<annotation id="392">
<infon key="score">0.9979323</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16784" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="973">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:38:45Z</infon>
<location offset="16805" length="22"/>
<text>hNaa60(1-199) F34A/CoA</text>
</annotation>
<annotation id="393">
<infon key="score">0.9976439</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16833" length="9"/>
<text>structure</text>
</annotation>
<annotation id="394">
<infon key="score">0.9936772</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:43:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16851" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="955">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<location offset="16879" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="395">
<infon key="score">0.99862635</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16931" length="16"/>
<text>electron density</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17026</offset>
<text>Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.</text>
<annotation id="396">
<infon key="score">0.99686694</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17026" length="6"/>
<text>Phe 27</text>
</annotation>
<annotation id="397">
<infon key="score">0.9991411</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17036" length="7"/>
<text>hNaa50p</text>
</annotation>
<annotation id="398">
<infon key="score">0.9948791</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17059" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="399">
<infon key="score">0.9993376</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17069" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="400">
<infon key="score">0.997621</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:24:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17137" length="10"/>
<text>methionine</text>
</annotation>
<annotation id="991">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="17165" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="401">
<infon key="score">0.996629</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17181" length="23"/>
<text>hydrophobic interaction</text>
</annotation>
<annotation id="402">
<infon key="score">0.9990492</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:41:12Z</infon>
<infon key="identifier">GO:</infon>
<location offset="17222" length="13"/>
<text>hNaa60/Ac-CoA</text>
</annotation>
<annotation id="403">
<infon key="score">0.99804497</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17236" length="9"/>
<text>structure</text>
</annotation>
<annotation id="404">
<infon key="score">0.9988341</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17261" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="405">
<infon key="score">0.9970541</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:24:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17330" length="10"/>
<text>methionine</text>
</annotation>
<annotation id="406">
<infon key="score">0.9985031</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17376" length="13"/>
<text>superposition</text>
</annotation>
<annotation id="407">
<infon key="score">0.9941642</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17417" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="408">
<infon key="score">0.9908397</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17504" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="409">
<infon key="score">0.99226713</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
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<infon key="updated_at">2023-09-20T09:47:40Z</infon>
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<text>enzyme kinetic data</text>
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<location offset="18083" length="13"/>
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<infon key="updated_at">2023-09-20T10:42:55Z</infon>
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<location offset="18097" length="4"/>
<text>F34A</text>
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<location offset="18290" length="18"/>
<text>circular dichroism</text>
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<location offset="18310" length="2"/>
<text>CD</text>
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<infon key="updated_at">2023-09-20T10:42:34Z</infon>
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<text>spectrum</text>
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<infon key="updated_at">2023-09-20T10:33:46Z</infon>
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<location offset="18367" length="17"/>
<text>crystal structure</text>
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<location offset="18425" length="4"/>
<text>F34A</text>
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<infon key="updated_at">2023-09-20T10:43:01Z</infon>
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<location offset="18430" length="6"/>
<text>mutant</text>
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<infon key="updated_at">2023-09-20T10:42:06Z</infon>
<location offset="18448" length="11"/>
<text>well-folded</text>
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<infon key="updated_at">2023-09-20T10:30:07Z</infon>
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<location offset="18511" length="10"/>
<text>α1-α2 loop</text>
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<infon key="updated_at">2023-09-20T09:47:40Z</infon>
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<text>Phe 34</text>
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<annotation id="992">
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<infon key="updated_at">2023-09-20T10:45:09Z</infon>
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<text>peptide</text>
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<infon key="updated_at">2023-09-20T10:40:53Z</infon>
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<location offset="18758" length="13"/>
<text>ethanethioate</text>
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<location offset="18782" length="6"/>
<text>Ac-CoA</text>
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<infon key="updated_at">2023-09-20T11:19:06Z</infon>
<location offset="18825" length="6"/>
<text>acetyl</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>18850</offset>
<text>Structural basis for hNaa60 substrate binding</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18871" length="6"/>
<text>hNaa60</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18896</offset>
<text>Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.</text>
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<infon key="score">0.99936503</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18966" length="6"/>
<text>hNaa60</text>
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<infon key="score">0.9993616</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18977" length="6"/>
<text>hNaa50</text>
</annotation>
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<infon key="score">0.99719715</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19011" length="9"/>
<text>structure</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:46:51Z</infon>
<infon key="identifier">GO:</infon>
<location offset="19024" length="19"/>
<text>hNaa50p/CoA/peptide</text>
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<annotation id="438">
<infon key="score">0.9990568</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19133" length="11"/>
<text>active site</text>
</annotation>
<annotation id="439">
<infon key="score">0.9993986</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19148" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="440">
<infon key="score">0.9990715</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19170" length="11"/>
<text>active site</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="19185" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
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<annotation id="442">
<infon key="score">0.99830693</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:13Z</infon>
<infon key="identifier">GO:</infon>
<location offset="19211" length="19"/>
<text>hNaa50p/CoA/peptide</text>
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<infon key="score">0.99739283</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19249" length="40"/>
<text>catalytic and substrate binding residues</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19294" length="16"/>
<text>highly conserved</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19366" length="7"/>
<text>hNaa50p</text>
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<infon key="score">0.9956695</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:22Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>Tyr 73</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:26Z</infon>
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<location offset="19419" length="7"/>
<text>His 112</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:41Z</infon>
<location offset="19514" length="12"/>
<text>well-ordered</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:55Z</infon>
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<location offset="19527" length="5"/>
<text>water</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:41Z</infon>
<location offset="19536" length="12"/>
<text>well-ordered</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19549" length="5"/>
<text>water</text>
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<annotation id="450">
<infon key="score">0.992056</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19578" length="6"/>
<text>Tyr 97</text>
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<infon key="score">0.9913372</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19589" length="7"/>
<text>His 138</text>
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<infon key="updated_at">2023-09-20T10:48:15Z</infon>
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<location offset="19600" length="18"/>
<text>hNaa60 (1-199)/CoA</text>
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<annotation id="453">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:18Z</infon>
<infon key="identifier">GO:</infon>
<location offset="19623" length="21"/>
<text>hNaa60 (1-242)/Ac-CoA</text>
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<annotation id="454">
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<infon key="updated_at">2023-09-20T09:47:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19685" length="6"/>
<text>Tyr 97</text>
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<annotation id="455">
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<infon key="updated_at">2023-09-20T09:48:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19696" length="7"/>
<text>His 138</text>
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<annotation id="456">
<infon key="score">0.9993262</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19707" length="6"/>
<text>hNaa60</text>
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<infon key="updated_at">2023-09-20T12:36:39Z</infon>
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<location offset="19728" length="7"/>
<text>mutated</text>
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<infon key="updated_at">2023-09-20T10:48:20Z</infon>
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<location offset="19754" length="7"/>
<text>alanine</text>
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<infon key="updated_at">2023-09-20T10:48:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19766" length="13"/>
<text>phenylalanine</text>
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<infon key="score">0.90800405</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19824" length="7"/>
<text>mutants</text>
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<infon key="score">0.9392303</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19844" length="14"/>
<text>kinetic assays</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:42:06Z</infon>
<location offset="19863" length="11"/>
<text>well-folded</text>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:42:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19878" length="2"/>
<text>CD</text>
</annotation>
<annotation id="463">
<infon key="score">0.95278716</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19881" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="464">
<infon key="score">0.9985738</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19945" length="8"/>
<text>SDS-PAGE</text>
</annotation>
<annotation id="465">
<infon key="score">0.99622065</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19991" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="466">
<infon key="score">0.998755</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19999" length="4"/>
<text>Y97A</text>
</annotation>
<annotation id="467">
<infon key="score">0.9989926</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20005" length="4"/>
<text>Y97F</text>
</annotation>
<annotation id="468">
<infon key="score">0.99900275</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20011" length="5"/>
<text>H138A</text>
</annotation>
<annotation id="469">
<infon key="score">0.9989298</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20021" length="5"/>
<text>H138F</text>
</annotation>
<annotation id="1050">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:37:26Z</infon>
<location offset="20027" length="22"/>
<text>abolished the activity</text>
</annotation>
<annotation id="470">
<infon key="score">0.9993457</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20053" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="471">
<infon key="score">0.9932173</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:36:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20077" length="6"/>
<text>mutate</text>
</annotation>
<annotation id="1049">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:36:57Z</infon>
<location offset="20095" length="15"/>
<text>solvent exposed</text>
</annotation>
<annotation id="472">
<infon key="score">0.9960622</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20119" length="6"/>
<text>Glu 37</text>
</annotation>
<annotation id="473">
<infon key="score">0.9937356</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:48:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20129" length="3"/>
<text>Ala</text>
</annotation>
<annotation id="474">
<infon key="score">0.9976242</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:34:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20134" length="4"/>
<text>E37A</text>
</annotation>
<annotation id="475">
<infon key="score">0.9993649</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20177" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="476">
<infon key="score">0.9985845</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20221" length="33"/>
<text>structural and functional studies</text>
</annotation>
<annotation id="477">
<infon key="score">0.9993759</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20269" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="478">
<infon key="score">0.9882264</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20320" length="6"/>
<text>Tyr 97</text>
</annotation>
<annotation id="479">
<infon key="score">0.9918355</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20328" length="7"/>
<text>His 138</text>
</annotation>
<annotation id="1004">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:41Z</infon>
<location offset="20342" length="12"/>
<text>well-ordered</text>
</annotation>
<annotation id="480">
<infon key="score">0.998632</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:47:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20355" length="5"/>
<text>water</text>
</annotation>
<annotation id="481">
<infon key="score">0.99939847</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20382" length="6"/>
<text>hNaa50</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>20394</offset>
<text>The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).</text>
<annotation id="482">
<infon key="score">0.99926704</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20398" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="483">
<infon key="score">0.99800014</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="20432" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="484">
<infon key="score">0.9984876</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20453" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="485">
<infon key="score">0.9993979</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20526" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="486">
<infon key="score">0.9989399</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20560" length="11"/>
<text>active site</text>
</annotation>
<annotation id="487">
<infon key="score">0.9947962</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20600" length="3"/>
<text>Met</text>
</annotation>
<annotation id="993">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="20621" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="488">
<infon key="score">0.9987845</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20636" length="13"/>
<text>superposition</text>
</annotation>
<annotation id="489">
<infon key="score">0.999078</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:41Z</infon>
<infon key="identifier">GO:</infon>
<location offset="20659" length="19"/>
<text>hNaa50p/CoA/peptide</text>
</annotation>
<annotation id="490">
<infon key="score">0.9886729</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20679" length="9"/>
<text>structure</text>
</annotation>
<annotation id="491">
<infon key="score">0.9944216</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20709" length="6"/>
<text>Tyr 38</text>
</annotation>
<annotation id="492">
<infon key="score">0.99544126</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20717" length="7"/>
<text>Asn 143</text>
</annotation>
<annotation id="493">
<infon key="score">0.99556684</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20729" length="7"/>
<text>Tyr 165</text>
</annotation>
<annotation id="494">
<infon key="score">0.9989592</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20760" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="495">
<infon key="score">0.99632376</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20805" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="496">
<infon key="score">0.9881353</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20823" length="12"/>
<text>water bridge</text>
</annotation>
<annotation id="497">
<infon key="score">0.9986852</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20856" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="498">
<infon key="score">0.9957231</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20920" length="6"/>
<text>lysine</text>
</annotation>
<annotation id="994">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="20938" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="499">
<infon key="score">0.9969008</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20972" length="24"/>
<text>hydrophilic interactions</text>
</annotation>
<annotation id="500">
<infon key="score">0.99480635</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21070" length="6"/>
<text>Tyr 38</text>
</annotation>
<annotation id="501">
<infon key="score">0.9955844</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21078" length="7"/>
<text>Asn 143</text>
</annotation>
<annotation id="502">
<infon key="score">0.9956566</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21090" length="7"/>
<text>Tyr 165</text>
</annotation>
<annotation id="503">
<infon key="score">0.99890196</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:34:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21197" length="4"/>
<text>Y38A</text>
</annotation>
<annotation id="504">
<infon key="score">0.99893147</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:34:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21203" length="5"/>
<text>N143A</text>
</annotation>
<annotation id="505">
<infon key="score">0.99892247</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:34:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21213" length="5"/>
<text>Y165A</text>
</annotation>
<annotation id="506">
<infon key="score">0.9975339</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21219" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="507">
<infon key="score">0.99915206</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21283" length="2"/>
<text>WT</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>21378</offset>
<text>The β3-β4 loop participates in the regulation of hNaa60-activity</text>
<annotation id="508">
<infon key="score">0.9993284</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21382" length="10"/>
<text>β3-β4 loop</text>
</annotation>
<annotation id="509">
<infon key="score">0.9993001</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21427" length="6"/>
<text>hNaa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>21449</offset>
<text>Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.</text>
<annotation id="510">
<infon key="score">0.99939644</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:00:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21466" length="2"/>
<text>β3</text>
</annotation>
<annotation id="511">
<infon key="score">0.9992778</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:00:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21473" length="2"/>
<text>β4</text>
</annotation>
<annotation id="512">
<infon key="score">0.99935776</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21479" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="1014">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:00:43Z</infon>
<location offset="21500" length="20"/>
<text>20-residue long loop</text>
</annotation>
<annotation id="513">
<infon key="score">0.99748373</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:00:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21531" length="5"/>
<text>73–92</text>
</annotation>
<annotation id="514">
<infon key="score">0.9959029</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21548" length="10"/>
<text>short turn</text>
</annotation>
<annotation id="899">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<location offset="21573" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="515">
<infon key="score">0.97910434</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:55:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21626" length="16"/>
<text>auto-acetylation</text>
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<annotation id="1008">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:55:49Z</infon>
<location offset="21646" length="6"/>
<text>hNaa60</text>
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<annotation id="1009">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:02Z</infon>
<location offset="21652" length="3"/>
<text>K79</text>
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<annotation id="516">
<infon key="score">0.9993579</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21688" length="6"/>
<text>hNaa60</text>
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<annotation id="517">
<infon key="score">0.99005854</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21738" length="6"/>
<text>Lys 79</text>
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<annotation id="518">
<infon key="score">0.9959693</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21748" length="10"/>
<text>acetylated</text>
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<annotation id="519">
<infon key="score">0.9988948</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21766" length="18"/>
<text>crystal structures</text>
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<annotation id="520">
<infon key="score">0.9986427</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21812" length="16"/>
<text>electron density</text>
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<annotation id="521">
<infon key="score">0.9889725</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21832" length="6"/>
<text>Lys 79</text>
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<annotation id="522">
<infon key="score">0.9986272</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:32:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21869" length="17"/>
<text>mass spectrometry</text>
</annotation>
<annotation id="523">
<infon key="score">0.98724186</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21901" length="6"/>
<text>Lys 79</text>
</annotation>
<annotation id="524">
<infon key="score">0.9929476</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21912" length="10"/>
<text>acetylated</text>
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<annotation id="1012">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:57:12Z</infon>
<location offset="22043" length="6"/>
<text>hNaa60</text>
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<annotation id="1013">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:57:25Z</infon>
<location offset="22049" length="3"/>
<text>K79</text>
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<annotation id="525">
<infon key="score">0.9744764</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22053" length="16"/>
<text>auto-acetylation</text>
</annotation>
<annotation id="526">
<infon key="score">0.9990439</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22098" length="4"/>
<text>K79R</text>
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<annotation id="527">
<infon key="score">0.99908864</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22107" length="4"/>
<text>K79Q</text>
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<annotation id="528">
<infon key="score">0.98770905</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22112" length="7"/>
<text>mutants</text>
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<annotation id="529">
<infon key="score">0.99886703</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22136" length="13"/>
<text>un-acetylated</text>
</annotation>
<annotation id="530">
<infon key="score">0.9990865</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22154" length="10"/>
<text>acetylated</text>
</annotation>
<annotation id="531">
<infon key="score">0.983596</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22173" length="6"/>
<text>Lys 79</text>
</annotation>
<annotation id="532">
<infon key="score">0.99891984</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22215" length="4"/>
<text>K79R</text>
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<annotation id="533">
<infon key="score">0.99896896</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22224" length="4"/>
<text>K79Q</text>
</annotation>
<annotation id="534">
<infon key="score">0.6879954</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22229" length="7"/>
<text>mutants</text>
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<annotation id="535">
<infon key="score">0.9993623</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22285" length="6"/>
<text>hNaa60</text>
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<annotation id="536">
<infon key="score">0.9991049</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22299" length="4"/>
<text>K79A</text>
</annotation>
<annotation id="537">
<infon key="score">0.96566397</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:43:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22304" length="6"/>
<text>mutant</text>
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<annotation id="538">
<infon key="score">0.99336725</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22390" length="11"/>
<text>acetylation</text>
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<annotation id="539">
<infon key="score">0.9828423</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22405" length="6"/>
<text>Lys 79</text>
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<annotation id="540">
<infon key="score">0.9993723</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22462" length="6"/>
<text>hNaa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>22485</offset>
<text>It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.</text>
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<infon key="score">0.999129</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22506" length="10"/>
<text>β3-β4 loop</text>
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<annotation id="542">
<infon key="score">0.9993956</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22520" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="543">
<infon key="score">0.8664518</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22569" length="25"/>
<text>substrate-binding pathway</text>
</annotation>
<annotation id="544">
<infon key="score">0.99904037</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22626" length="10"/>
<text>β3-β4 loop</text>
</annotation>
<annotation id="995">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="22674" length="7"/>
<text>peptide</text>
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<annotation id="1051">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:37:45Z</infon>
<location offset="22706" length="16"/>
<text>solvent-exposing</text>
</annotation>
<annotation id="545">
<infon key="score">0.995547</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22723" length="6"/>
<text>Lys 79</text>
</annotation>
<annotation id="546">
<infon key="score">0.9951457</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22833" length="6"/>
<text>Glu 80</text>
</annotation>
<annotation id="547">
<infon key="score">0.99640024</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22841" length="6"/>
<text>Asp 81</text>
</annotation>
<annotation id="548">
<infon key="score">0.9962824</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22852" length="6"/>
<text>Asp 83</text>
</annotation>
<annotation id="549">
<infon key="score">0.9954164</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22873" length="7"/>
<text>His 138</text>
</annotation>
<annotation id="550">
<infon key="score">0.99452215</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22882" length="7"/>
<text>His 159</text>
</annotation>
<annotation id="551">
<infon key="score">0.9952251</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22894" length="7"/>
<text>His 158</text>
</annotation>
<annotation id="552">
<infon key="score">0.99881583</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:50:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22938" length="10"/>
<text>β3-β4 loop</text>
</annotation>
<annotation id="553">
<infon key="score">0.96276665</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:37:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23040" length="7"/>
<text>mutated</text>
</annotation>
<annotation id="554">
<infon key="score">0.99578</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23048" length="6"/>
<text>Glu 80</text>
</annotation>
<annotation id="555">
<infon key="score">0.996125</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23056" length="6"/>
<text>Asp 81</text>
</annotation>
<annotation id="556">
<infon key="score">0.9965105</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:01:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23067" length="6"/>
<text>Asp 83</text>
</annotation>
<annotation id="557">
<infon key="score">0.99566793</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23077" length="3"/>
<text>Ala</text>
</annotation>
<annotation id="558">
<infon key="score">0.99891067</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23124" length="4"/>
<text>E80A</text>
</annotation>
<annotation id="559">
<infon key="score">0.9990312</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23130" length="4"/>
<text>D81A</text>
</annotation>
<annotation id="560">
<infon key="score">0.9990434</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23139" length="4"/>
<text>D83A</text>
</annotation>
<annotation id="561">
<infon key="score">0.9980446</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23144" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="562">
<infon key="score">0.99934214</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23188" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="563">
<infon key="score">0.998055</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23234" length="9"/>
<text>structure</text>
</annotation>
<annotation id="564">
<infon key="score">0.99928576</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23260" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="565">
<infon key="score">0.99852866</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:02:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23269" length="15"/>
<text>S. solfataricus</text>
</annotation>
<annotation id="1015">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:04:25Z</infon>
<location offset="23301" length="25"/>
<text>10-residue long extension</text>
</annotation>
<annotation id="566">
<infon key="score">0.99925405</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23335" length="2"/>
<text>β3</text>
</annotation>
<annotation id="567">
<infon key="score">0.9989618</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23342" length="2"/>
<text>β4</text>
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<annotation id="568">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:04:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23354" length="33"/>
<text>structure and biochemical studies</text>
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<infon key="score">0.9962972</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23404" length="9"/>
<text>extension</text>
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<infon key="score">0.9946806</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:03:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23417" length="5"/>
<text>SsNat</text>
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<annotation id="571">
<infon key="score">0.998391</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23469" length="11"/>
<text>active site</text>
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<infon key="score">0.9979418</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:03:56Z</infon>
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<location offset="23496" length="5"/>
<text>SsNat</text>
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</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>23536</offset>
<text>Discussion</text>
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<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>23547</offset>
<text>Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.</text>
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<infon key="score">0.9665944</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23547" length="14"/>
<text>Nt-acetylation</text>
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<annotation id="574">
<infon key="score">0.9985712</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:06:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23591" length="29"/>
<text>NAT family acetyltransferases</text>
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<annotation id="575">
<infon key="score">0.99907494</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23690" length="4"/>
<text>NATs</text>
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<annotation id="576">
<infon key="score">0.9988533</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:06:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23699" length="16"/>
<text>highly conserved</text>
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<annotation id="577">
<infon key="score">0.99409544</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:06:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23721" length="5"/>
<text>lower</text>
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<annotation id="578">
<infon key="score">0.94689333</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:06:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23730" length="17"/>
<text>higher eukaryotes</text>
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<annotation id="895">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:03Z</infon>
<location offset="23871" length="22"/>
<text>N-terminal acetylation</text>
</annotation>
<annotation id="579">
<infon key="score">0.9925706</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:06:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23899" length="5"/>
<text>lower</text>
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<annotation id="580">
<infon key="score">0.97923696</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:06:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23908" length="17"/>
<text>higher eukaryotes</text>
</annotation>
<annotation id="581">
<infon key="score">0.99926347</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23977" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="582">
<infon key="score">0.9782407</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23997" length="24"/>
<text>multicellular eukaryotes</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>24023</offset>
<text>The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.</text>
<annotation id="583">
<infon key="score">0.9993436</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24041" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="584">
<infon key="score">0.32769075</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24084" length="3"/>
<text>VVP</text>
</annotation>
<annotation id="585">
<infon key="score">0.99827373</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:38:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24180" length="9"/>
<text>replacing</text>
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<annotation id="586">
<infon key="score">0.99464613</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:33:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24199" length="3"/>
<text>4–6</text>
</annotation>
<annotation id="587">
<infon key="score">0.39737746</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24208" length="3"/>
<text>VVP</text>
</annotation>
<annotation id="588">
<infon key="score">0.3354909</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24215" length="3"/>
<text>EER</text>
</annotation>
<annotation id="589">
<infon key="score">0.9994191</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24237" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="590">
<infon key="score">0.99843544</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24248" length="14"/>
<text>Xenopus Laevis</text>
</annotation>
<annotation id="591">
<infon key="score">0.9994129</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24270" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="592">
<infon key="score">0.9987526</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24281" length="5"/>
<text>human</text>
</annotation>
<annotation id="593">
<infon key="score">0.9984007</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24296" length="14"/>
<text>Xenopus Laevis</text>
</annotation>
<annotation id="594">
<infon key="score">0.655985</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24315" length="17"/>
<text>highly homologous</text>
</annotation>
<annotation id="595">
<infon key="score">0.6868272</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:05:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24456" length="10"/>
<text>VVP to EER</text>
</annotation>
<annotation id="596">
<infon key="score">0.99554366</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:05:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24467" length="11"/>
<text>replacement</text>
</annotation>
<annotation id="597">
<infon key="score">0.9993748</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24500" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="908">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<location offset="24560" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="964">
<infon key="type">residue_range</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<location offset="24567" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="598">
<infon key="score">0.9979784</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24574" length="9"/>
<text>structure</text>
</annotation>
<annotation id="599">
<infon key="score">0.9973535</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24609" length="19"/>
<text>α-helical structure</text>
</annotation>
<annotation id="600">
<infon key="score">0.9877457</infon>
<infon key="type">residue_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24670" length="1"/>
<text>6</text>
</annotation>
<annotation id="601">
<infon key="score">0.9979997</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24675" length="7"/>
<text>proline</text>
</annotation>
<annotation id="602">
<infon key="score">0.95973444</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24705" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="603">
<infon key="score">0.9979778</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24719" length="9"/>
<text>structure</text>
</annotation>
<annotation id="604">
<infon key="score">0.9946813</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24763" length="22"/>
<text>semi-helical structure</text>
</annotation>
<annotation id="981">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:44:17Z</infon>
<location offset="24820" length="15"/>
<text>crystal packing</text>
</annotation>
<annotation id="605">
<infon key="score">0.9991526</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24884" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="606">
<infon key="score">0.9993598</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24894" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="607">
<infon key="score">0.9992042</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:30:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24907" length="7"/>
<text>α-helix</text>
</annotation>
<annotation id="608">
<infon key="score">0.9663139</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:08:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24956" length="3"/>
<text>4–6</text>
</annotation>
<annotation id="609">
<infon key="score">0.9991584</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24994" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="610">
<infon key="score">0.9993875</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25004" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="611">
<infon key="score">0.5227871</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25045" length="8"/>
<text>mutation</text>
</annotation>
<annotation id="612">
<infon key="score">0.4418587</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25055" length="3"/>
<text>VVP</text>
</annotation>
<annotation id="613">
<infon key="score">0.51431394</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:19:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25062" length="3"/>
<text>EER</text>
</annotation>
<annotation id="614">
<infon key="score">0.99928135</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:46Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25093" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="615">
<infon key="score">0.99907476</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25125" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="616">
<infon key="score">0.986394</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25153" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="617">
<infon key="score">0.9944792</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:08:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25171" length="5"/>
<text>1-199</text>
</annotation>
<annotation id="618">
<infon key="score">0.959848</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25234" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="619">
<infon key="score">0.9730511</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25241" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="1046">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:34:58Z</infon>
<location offset="25275" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="620">
<infon key="score">0.9990978</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25342" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="621">
<infon key="score">0.99933344</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25354" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="622">
<infon key="score">0.9991409</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25515" length="11"/>
<text>full-length</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>25544</offset>
<text>The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies.</text>
<annotation id="623">
<infon key="score">0.9991893</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25548" length="6"/>
<text>hNaa60</text>
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<annotation id="624">
<infon key="score">0.99866015</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25651" length="21"/>
<text>transmembrane domains</text>
</annotation>
<annotation id="625">
<infon key="score">0.97566384</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25699" length="16"/>
<text>S-palmitoylation</text>
</annotation>
<annotation id="626">
<infon key="score">0.99236155</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:38:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25802" length="7"/>
<text>mutated</text>
</annotation>
<annotation id="627">
<infon key="score">0.9971693</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:08:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25819" length="8"/>
<text>cysteine</text>
</annotation>
<annotation id="628">
<infon key="score">0.9992047</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25840" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="629">
<infon key="score">0.9971943</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:08:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25852" length="6"/>
<text>serine</text>
</annotation>
<annotation id="630">
<infon key="score">0.99831057</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25887" length="22"/>
<text>S-palmitoylation sites</text>
</annotation>
<annotation id="631">
<infon key="score">0.97516257</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:38:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25926" length="9"/>
<text>mutations</text>
</annotation>
<annotation id="632">
<infon key="score">0.99930024</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25952" length="5"/>
<text>Naa60</text>
</annotation>
<annotation id="633">
<infon key="score">0.9770059</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25997" length="16"/>
<text>S-palmitoylation</text>
</annotation>
<annotation id="634">
<infon key="score">0.99929214</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26060" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="635">
<infon key="score">0.9947547</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:38:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26090" length="6"/>
<text>adding</text>
</annotation>
<annotation id="636">
<infon key="score">0.9976034</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:08:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26106" length="7"/>
<text>217–242</text>
</annotation>
<annotation id="637">
<infon key="score">0.9992981</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26117" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="638">
<infon key="score">0.9976539</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26145" length="7"/>
<text>217–236</text>
</annotation>
<annotation id="639">
<infon key="score">0.997695</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:09:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26174" length="21"/>
<text>transmembrane domains</text>
</annotation>
<annotation id="640">
<infon key="score">0.92742413</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:09:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26218" length="4"/>
<text>eGFP</text>
</annotation>
<annotation id="641">
<infon key="score">0.7607362</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:23:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26293" length="4"/>
<text>eGFP</text>
</annotation>
<annotation id="642">
<infon key="score">0.8553622</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:35:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26298" length="13"/>
<text>hNaa60182-242</text>
</annotation>
<annotation id="643">
<infon key="score">0.9976466</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26356" length="7"/>
<text>182–216</text>
</annotation>
<annotation id="644">
<infon key="score">0.9992848</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26404" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="645">
<infon key="score">0.99767846</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:10:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26435" length="7"/>
<text>190–202</text>
</annotation>
<annotation id="646">
<infon key="score">0.97816676</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26453" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="1016">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:11:02Z</infon>
<location offset="26606" length="19"/>
<text>amphipathic helices</text>
</annotation>
<annotation id="647">
<infon key="score">0.9991417</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:53Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26642" length="8"/>
<text>KalSec14</text>
</annotation>
<annotation id="648">
<infon key="score">0.9991431</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:28:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26652" length="4"/>
<text>Atg3</text>
</annotation>
<annotation id="649">
<infon key="score">0.999237</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:04Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26658" length="6"/>
<text>PB1-F2</text>
</annotation>
<annotation id="969">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:48Z</infon>
<location offset="26687" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="650">
<infon key="score">0.99526036</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:26:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26769" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
<annotation id="970">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:48Z</infon>
<location offset="26825" length="17"/>
<text>amphipathic helix</text>
</annotation>
<annotation id="651">
<infon key="score">0.99937785</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:31:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26843" length="2"/>
<text>α5</text>
</annotation>
<annotation id="652">
<infon key="score">0.99927133</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26886" length="6"/>
<text>hNaa60</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>26986</offset>
<text>Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.</text>
<annotation id="900">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<location offset="27029" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="653">
<infon key="score">0.99865484</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27058" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="654">
<infon key="score">0.9953199</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27066" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="655">
<infon key="score">0.99714917</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:11:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27094" length="10"/>
<text>structures</text>
</annotation>
<annotation id="656">
<infon key="score">0.9986947</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27108" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="657">
<infon key="score">0.9985568</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:31:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27160" length="13"/>
<text>β6-β7 hairpin</text>
</annotation>
<annotation id="658">
<infon key="score">0.99843925</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27174" length="4"/>
<text>loop</text>
</annotation>
<annotation id="659">
<infon key="score">0.9988381</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27190" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="1052">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:50Z</infon>
<location offset="27229" length="34"/>
<text>tunnel-like substrate-binding site</text>
</annotation>
<annotation id="660">
<infon key="score">0.99911714</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27273" length="10"/>
<text>α1-α2 loop</text>
</annotation>
<annotation id="661">
<infon key="score">0.995466</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27313" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="662">
<infon key="score">0.9779108</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27325" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="663">
<infon key="score">0.9656652</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27353" length="15"/>
<text>Kinetic studies</text>
</annotation>
<annotation id="664">
<infon key="score">0.9873094</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27404" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="665">
<infon key="score">0.96045804</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27412" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="666">
<infon key="score">0.9993749</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27428" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="667">
<infon key="score">0.97203314</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27467" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="668">
<infon key="score">0.99943024</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:21:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27483" length="5"/>
<text>Naa50</text>
</annotation>
<annotation id="669">
<infon key="score">0.9692684</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27509" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="670">
<infon key="score">0.71763927</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27579" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="996">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="27604" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="1040">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:21Z</infon>
<location offset="27645" length="12"/>
<text>3D structure</text>
</annotation>
<annotation id="671">
<infon key="score">0.9810611</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27663" length="6"/>
<text>folded</text>
</annotation>
<annotation id="672">
<infon key="score">0.9983854</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:32:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27701" length="17"/>
<text>mass spectrometry</text>
</annotation>
<annotation id="1041">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:39Z</infon>
<location offset="27719" length="4"/>
<text>data</text>
</annotation>
<annotation id="673">
<infon key="score">0.99387944</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27757" length="11"/>
<text>acetylation</text>
</annotation>
<annotation id="923">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:08:43Z</infon>
<location offset="27772" length="7"/>
<text>histone</text>
</annotation>
<annotation id="972">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:32:13Z</infon>
<location offset="27780" length="5"/>
<text>H3-H4</text>
</annotation>
<annotation id="674">
<infon key="score">0.9655266</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27786" length="8"/>
<text>tetramer</text>
</annotation>
<annotation id="675">
<infon key="score">0.992084</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:24:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27795" length="7"/>
<text>lysines</text>
</annotation>
<annotation id="896">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:03Z</infon>
<location offset="27812" length="22"/>
<text>N-terminal acetylation</text>
</annotation>
<annotation id="918">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:05:37Z</infon>
<location offset="27839" length="18"/>
<text>lysine acetylation</text>
</annotation>
<annotation id="997">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="27865" length="7"/>
<text>peptide</text>
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<annotation id="676">
<infon key="score">0.99767715</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27885" length="14"/>
<text>activity assay</text>
</annotation>
<annotation id="677">
<infon key="score">0.97320694</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27920" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="678">
<infon key="score">0.9989168</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27987" length="13"/>
<text>β7-β8 hairpin</text>
</annotation>
<annotation id="679">
<infon key="score">0.99842685</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28023" length="10"/>
<text>β6-β7 loop</text>
</annotation>
<annotation id="680">
<infon key="score">0.9988734</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28043" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="681">
<infon key="score">0.99845386</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:11:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28065" length="10"/>
<text>structures</text>
</annotation>
<annotation id="682">
<infon key="score">0.99693537</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28136" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="683">
<infon key="score">0.9884763</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28140" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="684">
<infon key="score">0.99828833</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:12:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28286" length="7"/>
<text>hairpin</text>
</annotation>
<annotation id="982">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:44:17Z</infon>
<location offset="28326" length="15"/>
<text>crystal packing</text>
</annotation>
<annotation id="685">
<infon key="score">0.97712255</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28463" length="3"/>
<text>KAT</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>28518</offset>
<text>The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.</text>
<annotation id="1017">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:14:32Z</infon>
<location offset="28663" length="30"/>
<text>GCN5 histone acetyltransferase</text>
</annotation>
<annotation id="686">
<infon key="score">0.99628496</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:14:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28694" length="9"/>
<text>structure</text>
</annotation>
<annotation id="687">
<infon key="score">0.9992446</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28718" length="3"/>
<text>CoA</text>
</annotation>
<annotation id="688">
<infon key="score">0.9990343</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28736" length="9"/>
<text>complexed</text>
</annotation>
<annotation id="689">
<infon key="score">0.996225</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:54:46Z</infon>
<infon key="identifier">GO:</infon>
<location offset="28754" length="4"/>
<text>NatA</text>
</annotation>
<annotation id="690">
<infon key="score">0.9991602</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28812" length="10"/>
<text>α1-α2 loop</text>
</annotation>
<annotation id="691">
<infon key="score">0.9989286</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28890" length="22"/>
<text>substrate-binding site</text>
</annotation>
<annotation id="692">
<infon key="score">0.9994199</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:21:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28940" length="5"/>
<text>Naa15</text>
</annotation>
<annotation id="693">
<infon key="score">0.99938476</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:21:17Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28961" length="5"/>
<text>Naa10</text>
</annotation>
<annotation id="694">
<infon key="score">0.74719286</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:28:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28972" length="9"/>
<text>catalytic</text>
</annotation>
<annotation id="695">
<infon key="score">0.6046873</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:31:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28982" length="7"/>
<text>subunit</text>
</annotation>
<annotation id="696">
<infon key="score">0.97488165</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:54:46Z</infon>
<infon key="identifier">GO:</infon>
<location offset="28993" length="4"/>
<text>NatA</text>
</annotation>
<annotation id="697">
<infon key="score">0.99712</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:14:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29007" length="9"/>
<text>structure</text>
</annotation>
<annotation id="698">
<infon key="score">0.99884164</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:15:05Z</infon>
<infon key="identifier">GO:</infon>
<location offset="29020" length="18"/>
<text>hNaa50/CoA/peptide</text>
</annotation>
<annotation id="699">
<infon key="score">0.9969653</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29040" length="6"/>
<text>Phe 27</text>
</annotation>
<annotation id="700">
<infon key="score">0.9991973</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29054" length="10"/>
<text>α1-α2 loop</text>
</annotation>
<annotation id="701">
<infon key="score">0.9969026</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29081" length="23"/>
<text>hydrophobic interaction</text>
</annotation>
<annotation id="702">
<infon key="score">0.9954798</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:16:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29125" length="3"/>
<text>Met</text>
</annotation>
<annotation id="998">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:09Z</infon>
<location offset="29142" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="948">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<location offset="29164" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="703">
<infon key="score">0.99837536</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29185" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="704">
<infon key="score">0.9994266</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29237" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="705">
<infon key="score">0.9955821</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29245" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="706">
<infon key="score">0.99918216</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29305" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="707">
<infon key="score">0.9986929</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29332" length="22"/>
<text>substrate binding site</text>
</annotation>
<annotation id="708">
<infon key="score">0.99944645</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29420" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="709">
<infon key="score">0.6209677</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:23:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29456" length="5"/>
<text>thiol</text>
</annotation>
<annotation id="710">
<infon key="score">0.99915993</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29465" length="3"/>
<text>CoA</text>
</annotation>
<annotation id="711">
<infon key="score">0.99825984</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29510" length="9"/>
<text>structure</text>
</annotation>
<annotation id="712">
<infon key="score">0.9801244</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<infon key="identifier">GO:</infon>
<location offset="29523" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="713">
<infon key="score">0.99917614</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:30:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29613" length="10"/>
<text>α1-α2 loop</text>
</annotation>
<annotation id="714">
<infon key="score">0.9990846</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29693" length="3"/>
<text>CoA</text>
</annotation>
<annotation id="1018">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:16:09Z</infon>
<location offset="29718" length="19"/>
<text>hNaa60(1-199)(F34A)</text>
</annotation>
<annotation id="715">
<infon key="score">0.998599</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29738" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="716">
<infon key="score">0.97129583</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:25:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29765" length="12"/>
<text>kinetic data</text>
</annotation>
<annotation id="717">
<infon key="score">0.9991026</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:42:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29794" length="4"/>
<text>F34A</text>
</annotation>
<annotation id="943">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:07Z</infon>
<location offset="29799" length="8"/>
<text>mutation</text>
</annotation>
<annotation id="718">
<infon key="score">0.9954623</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29886" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="719">
<infon key="score">0.9994444</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29896" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="1020">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:19:05Z</infon>
<location offset="29980" length="6"/>
<text>acetyl</text>
</annotation>
<annotation id="720">
<infon key="score">0.99600923</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30056" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="721">
<infon key="score">0.99463564</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:16:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30108" length="3"/>
<text>Met</text>
</annotation>
<annotation id="722">
<infon key="score">0.99646735</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:27:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30120" length="23"/>
<text>hydrophobic interaction</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>30205</offset>
<text>Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.</text>
<annotation id="723">
<infon key="score">0.9992888</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30233" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="1024">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:19:06Z</infon>
<location offset="30292" length="6"/>
<text>acetyl</text>
</annotation>
<annotation id="724">
<infon key="score">0.9992924</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30344" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="725">
<infon key="score">0.9993185</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30355" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="726">
<infon key="score">0.9990032</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30390" length="11"/>
<text>active site</text>
</annotation>
<annotation id="727">
<infon key="score">0.99891543</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:29:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30422" length="16"/>
<text>highly conserved</text>
</annotation>
<annotation id="728">
<infon key="score">0.99930453</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30522" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="729">
<infon key="score">0.9993167</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30533" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="730">
<infon key="score">0.99931</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30571" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="731">
<infon key="score">0.9991873</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30611" length="4"/>
<text>NATs</text>
</annotation>
<annotation id="732">
<infon key="score">0.99923515</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30623" length="15"/>
<text>β3-β4 long loop</text>
</annotation>
<annotation id="733">
<infon key="score">0.9992981</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30690" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="734">
<infon key="score">0.9965564</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:18:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30712" length="4"/>
<text>loop</text>
</annotation>
<annotation id="735">
<infon key="score">0.99934274</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30751" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="736">
<infon key="score">0.9930085</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:26:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30758" length="9"/>
<text>structure</text>
</annotation>
<annotation id="737">
<infon key="score">0.9984491</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:38:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30777" length="18"/>
<text>deletion mutations</text>
</annotation>
<annotation id="738">
<infon key="score">0.9753645</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:18:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30905" length="16"/>
<text>auto-acetylation</text>
</annotation>
<annotation id="739">
<infon key="score">0.98703116</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30925" length="6"/>
<text>Lys 79</text>
</annotation>
<annotation id="740">
<infon key="score">0.9992586</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30950" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="741">
<infon key="score">0.879584</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:17:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30979" length="14"/>
<text>point mutation</text>
</annotation>
<annotation id="742">
<infon key="score">0.99910057</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30994" length="4"/>
<text>K79R</text>
</annotation>
<annotation id="743">
<infon key="score">0.99931717</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31032" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="744">
<infon key="score">0.9931169</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31067" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="1025">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:19:06Z</infon>
<location offset="31087" length="6"/>
<text>acetyl</text>
</annotation>
<annotation id="745">
<infon key="score">0.9799822</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31113" length="6"/>
<text>Lys 79</text>
</annotation>
<annotation id="746">
<infon key="score">0.9984842</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:26:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31127" length="10"/>
<text>structures</text>
</annotation>
<annotation id="747">
<infon key="score">0.9960762</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:32:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31142" length="17"/>
<text>mass spectrometry</text>
</annotation>
<annotation id="748">
<infon key="score">0.97629786</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:18:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31197" length="16"/>
<text>auto-acetylation</text>
</annotation>
<annotation id="749">
<infon key="score">0.9993073</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31217" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="750">
<infon key="score">0.9990808</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31326" length="4"/>
<text>K79R</text>
</annotation>
<annotation id="751">
<infon key="score">0.9982279</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:43:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31462" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="752">
<infon key="score">0.99911886</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:56:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31489" length="4"/>
<text>K79R</text>
</annotation>
<annotation id="753">
<infon key="score">0.89987457</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:18:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31502" length="6"/>
<text>stable</text>
</annotation>
<annotation id="754">
<infon key="score">0.9989593</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:07:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31518" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1019">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:17:54Z</infon>
<location offset="31563" length="14"/>
<text>gel-filtration</text>
</annotation>
<annotation id="1007">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:49:16Z</infon>
<location offset="31767" length="14"/>
<text>kinetic assays</text>
</annotation>
<annotation id="755">
<infon key="score">0.99931145</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31890" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="756">
<infon key="score">0.99931645</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31961" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="757">
<infon key="score">0.9949426</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:18:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32100" length="9"/>
<text>structure</text>
</annotation>
<annotation id="758">
<infon key="score">0.9992893</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:47Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32113" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="901">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<location offset="32344" length="3"/>
<text>NAT</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>32362</offset>
<text>Methods</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32370</offset>
<text>Cloning, expression and purification of Homo sapiens Naa60 (hNaa60)</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32438</offset>
<text>The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>34517</offset>
<text>Circular Dichroism (CD) Spectroscopy</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34554</offset>
<text>CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>34949</offset>
<text>Crystallization, data collection and structure determination</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>35010</offset>
<text>The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>35814</offset>
<text>The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>36891</offset>
<text>Acetyltransferase assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>36915</offset>
<text>Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1).</text>
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<text>Author Contributions J.-Y.C., L.L., C.-L.C., M.-J.L. and X.Y. designed and performed the experiments. K.T. collected diffraction data at APS. C.-H.Y. conceived and instructed the project. All authors are involved in data analysis. J.-Y.C. and C.-H.Y. wrote the manuscript.</text>
</passage>
<passage>
<infon key="file">srep31425-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>42521</offset>
<text>Overall structure of Naa60.</text>
<annotation id="759">
<infon key="score">0.99692875</infon>
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<infon key="updated_at">2023-09-20T12:07:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42529" length="9"/>
<text>structure</text>
</annotation>
<annotation id="760">
<infon key="score">0.9992374</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42542" length="5"/>
<text>Naa60</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31425-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>42549</offset>
<text>(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.</text>
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<infon key="score">0.9933519</infon>
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<infon key="updated_at">2023-09-20T12:07:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42553" length="18"/>
<text>Sequence alignment</text>
</annotation>
<annotation id="762">
<infon key="score">0.9992231</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:44:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42575" length="5"/>
<text>Naa60</text>
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<annotation id="763">
<infon key="score">0.874855</infon>
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<infon key="updated_at">2023-09-20T09:55:36Z</infon>
<infon key="identifier">GO:</infon>
<location offset="42582" length="4"/>
<text>NatF</text>
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<infon key="score">0.7399594</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:03:19Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42588" length="4"/>
<text>HAT4</text>
</annotation>
<annotation id="765">
<infon key="score">0.99795175</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:20:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42627" length="12"/>
<text>Homo sapiens</text>
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<annotation id="766">
<infon key="score">0.99839276</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42641" length="4"/>
<text>Homo</text>
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<annotation id="767">
<infon key="score">0.9980613</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42648" length="9"/>
<text>Bos mutus</text>
</annotation>
<annotation id="768">
<infon key="score">0.9981675</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42659" length="3"/>
<text>Bos</text>
</annotation>
<annotation id="769">
<infon key="score">0.99803877</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42665" length="11"/>
<text>Salmo salar</text>
</annotation>
<annotation id="770">
<infon key="score">0.99874157</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42678" length="5"/>
<text>Salmo</text>
</annotation>
<annotation id="771">
<infon key="score">0.998781</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42689" length="7"/>
<text>Xenopus</text>
</annotation>
<annotation id="772">
<infon key="score">0.9966102</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42698" length="8"/>
<text>Silurana</text>
</annotation>
<annotation id="773">
<infon key="score">0.99740475</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:07:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42708" length="10"/>
<text>tropicalis</text>
</annotation>
<annotation id="774">
<infon key="score">0.9978409</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42720" length="7"/>
<text>Xenopus</text>
</annotation>
<annotation id="775">
<infon key="score">0.89359903</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42730" length="9"/>
<text>Alignment</text>
</annotation>
<annotation id="776">
<infon key="score">0.99120206</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42833" length="3"/>
<text>4–6</text>
</annotation>
<annotation id="777">
<infon key="score">0.9879758</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42873" length="9"/>
<text>structure</text>
</annotation>
<annotation id="956">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<location offset="42886" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="778">
<infon key="score">0.99892956</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="42952" length="3"/>
<text>CoA</text>
</annotation>
<annotation id="779">
<infon key="score">0.99506253</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42993" length="9"/>
<text>structure</text>
</annotation>
<annotation id="780">
<infon key="score">0.9562601</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="43006" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="781">
<infon key="score">0.99923086</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="43080" length="6"/>
<text>Ac-CoA</text>
</annotation>
<annotation id="782">
<infon key="score">0.9993051</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:40:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="43091" length="8"/>
<text>malonate</text>
</annotation>
<annotation id="783">
<infon key="score">0.9983317</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:08:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43212" length="2"/>
<text>α0</text>
</annotation>
<annotation id="784">
<infon key="score">0.9985247</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="43220" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="785">
<infon key="score">0.73757434</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43237" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="786">
<infon key="score">0.87932587</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43244" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="1027">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:09:15Z</infon>
<location offset="43259" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="787">
<infon key="score">0.99920577</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43286" length="6"/>
<text>hNaa50</text>
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<annotation id="788">
<infon key="score">0.99919695</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="43315" length="6"/>
<text>Ac-CoA</text>
</annotation>
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<infon key="score">0.9380579</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<infon key="identifier">GO:</infon>
<location offset="43325" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31425-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>43389</offset>
<text>Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.</text>
<annotation id="790">
<infon key="score">0.8762618</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:29:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43389" length="14"/>
<text>Amphipathicity</text>
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<annotation id="791">
<infon key="score">0.99929076</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:09:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43411" length="8"/>
<text>α5 helix</text>
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<annotation id="792">
<infon key="score">0.99915457</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43457" length="13"/>
<text>β7-β8 hairpin</text>
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<passage>
<infon key="file">srep31425-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>43482</offset>
<text>(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.</text>
<annotation id="793">
<infon key="score">0.99926305</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:09:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43490" length="8"/>
<text>α5 helix</text>
</annotation>
<annotation id="794">
<infon key="score">0.63434005</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43502" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="795">
<infon key="score">0.9174269</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43509" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="796">
<infon key="score">0.9991447</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43570" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="797">
<infon key="score">0.9989952</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:09:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43719" length="8"/>
<text>α5 helix</text>
</annotation>
<annotation id="798">
<infon key="score">0.99929494</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:09:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43850" length="8"/>
<text>α5 helix</text>
</annotation>
<annotation id="1028">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:10:20Z</infon>
<location offset="43862" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="799">
<infon key="score">0.9989097</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43931" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="800">
<infon key="score">0.9990158</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="44088" length="8"/>
<text>α5 helix</text>
</annotation>
<annotation id="801">
<infon key="score">0.99918485</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="44281" length="8"/>
<text>α5 helix</text>
</annotation>
<annotation id="802">
<infon key="score">0.9965055</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44295" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="1029">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:01Z</infon>
<location offset="44312" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="803">
<infon key="score">0.97378564</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44339" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="804">
<infon key="score">0.98355436</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44346" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="805">
<infon key="score">0.9991952</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="44397" length="13"/>
<text>β7-β8 hairpin</text>
</annotation>
<annotation id="806">
<infon key="score">0.99780375</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44424" length="10"/>
<text>structures</text>
</annotation>
<annotation id="807">
<infon key="score">0.9980095</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:29:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44442" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="808">
<infon key="score">0.998798</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44459" length="5"/>
<text>Hat1p</text>
</annotation>
<annotation id="809">
<infon key="score">0.9936947</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:09:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44465" length="2"/>
<text>H4</text>
</annotation>
<annotation id="810">
<infon key="score">0.994859</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44501" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="811">
<infon key="score">0.8863415</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44508" length="5"/>
<text>1-242</text>
</annotation>
<annotation id="1030">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:20Z</infon>
<location offset="44528" length="13"/>
<text>hNaa60(1-199)</text>
</annotation>
<annotation id="924">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:08:43Z</infon>
<location offset="44559" length="7"/>
<text>histone</text>
</annotation>
<annotation id="812">
<infon key="score">0.9862388</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:09:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44567" length="2"/>
<text>H4</text>
</annotation>
<annotation id="999">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:10Z</infon>
<location offset="44570" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="813">
<infon key="score">0.9983368</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44581" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="814">
<infon key="score">0.99917674</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44596" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="815">
<infon key="score">0.9994037</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44605" length="5"/>
<text>Hat1p</text>
</annotation>
<annotation id="1000">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:10Z</infon>
<location offset="44647" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="816">
<infon key="score">0.9989772</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44655" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="817">
<infon key="score">0.99940395</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44664" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="818">
<infon key="score">0.9982008</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44674" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="1036">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:23:52Z</infon>
<location offset="44730" length="10"/>
<text>Nt-peptide</text>
</annotation>
<annotation id="819">
<infon key="score">0.99784434</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44748" length="13"/>
<text>superimposing</text>
</annotation>
<annotation id="820">
<infon key="score">0.99932384</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44762" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="821">
<infon key="score">0.999337</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44794" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="822">
<infon key="score">0.9966156</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44825" length="3"/>
<text>NAT</text>
</annotation>
<annotation id="823">
<infon key="score">0.9981242</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44858" length="3"/>
<text>KAT</text>
</annotation>
<annotation id="824">
<infon key="score">0.9917366</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:11:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="44888" length="6"/>
<text>lysine</text>
</annotation>
<annotation id="825">
<infon key="score">0.9904163</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:48:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44918" length="11"/>
<text>acetylation</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31425-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>44987</offset>
<text>Electron density map of the active site.</text>
<annotation id="826">
<infon key="score">0.9986318</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:12:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44987" length="20"/>
<text>Electron density map</text>
</annotation>
<annotation id="827">
<infon key="score">0.9990533</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:34:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="45015" length="11"/>
<text>active site</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31425-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>45028</offset>
<text>The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).</text>
<annotation id="828">
<infon key="score">0.99838984</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:12:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45032" length="11"/>
<text>2Fo-Fc maps</text>
</annotation>
<annotation id="949">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:35Z</infon>
<location offset="45076" length="20"/>
<text>hNaa60(1-242)/Ac-CoA</text>
</annotation>
<annotation id="957">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:21:54Z</infon>
<location offset="45102" length="17"/>
<text>hNaa60(1-199)/CoA</text>
</annotation>
<annotation id="1031">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:12:54Z</infon>
<location offset="45128" length="22"/>
<text>hNaa60(1-199) F34A/CoA</text>
</annotation>
<annotation id="829">
<infon key="score">0.99883485</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:13:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="45169" length="30"/>
<text>substrate peptide binding site</text>
</annotation>
<annotation id="830">
<infon key="score">0.7129295</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T10:45:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="45220" length="7"/>
<text>peptide</text>
</annotation>
<annotation id="831">
<infon key="score">0.99902344</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:13:05Z</infon>
<infon key="identifier">GO:</infon>
<location offset="45260" length="18"/>
<text>hNaa50/CoA/peptide</text>
</annotation>
<annotation id="832">
<infon key="score">0.930311</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:13:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45287" length="9"/>
<text>structure</text>
</annotation>
<annotation id="833">
<infon key="score">0.9982256</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45303" length="13"/>
<text>superimposing</text>
</annotation>
<annotation id="834">
<infon key="score">0.99938524</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:20:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="45317" length="6"/>
<text>hNaa50</text>
</annotation>
<annotation id="835">
<infon key="score">0.9993223</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:46:48Z</infon>
<infon key="identifier">PR:</infon>
<location offset="45331" length="6"/>
<text>hNaa60</text>
</annotation>
<annotation id="836">
<infon key="score">0.9975768</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:26:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45338" length="10"/>
<text>structures</text>
</annotation>
<annotation id="1032">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:13:44Z</infon>
<location offset="45420" length="9"/>
<text>first Met</text>
</annotation>
<annotation id="837">
<infon key="score">0.99554753</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:13:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45431" length="2"/>
<text>M1</text>
</annotation>
<annotation id="1026">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T11:19:06Z</infon>
<location offset="45480" length="6"/>
<text>acetyl</text>
</annotation>
<annotation id="838">
<infon key="score">0.9990597</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="45497" length="6"/>
<text>Ac-CoA</text>
</annotation>
<annotation id="839">
<infon key="score">0.9792496</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T09:47:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="45604" length="6"/>
<text>Phe 34</text>
</annotation>
<annotation id="840">
<infon key="score">0.99492764</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:39:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45642" length="7"/>
<text>mutated</text>
</annotation>
<annotation id="841">
<infon key="score">0.9958605</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-20T12:15:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="45653" length="3"/>
<text>Ala</text>
</annotation>
</passage>
<passage>
<infon key="file">srep31425-f4.jpg</infon>
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<text>Structural basis for hNaa60 catalytic activity.</text>
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<text>(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges.</text>
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<text>Catalytic activity of hNaa60 and mutant proteins.</text>
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<text>(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants. Error bars indicate the Standard Error (SE). (B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm. The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature.</text>
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<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Structure and PDB ID</th><th align="center" valign="top" charoff="50">hNaa60(1-242)/Ac-CoA 5HGZ</th><th align="center" valign="top" charoff="50">hNaa60(1-199)/CoA 5HH0</th><th align="center" valign="top" charoff="50">hNaa60(1-199)F34A/CoA 5HH1</th></tr></thead><tbody valign="top"><tr><td colspan="4" align="left" valign="top" charoff="50">Data collection<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub></td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic></td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic></td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>a, b, c</italic> (Å)</td><td align="center" valign="top" charoff="50">53.3, 57.4, 68.8</td><td align="center" valign="top" charoff="50">67.8, 73.8, 43.2</td><td align="center" valign="top" charoff="50">66.7, 74.0, 43.5</td></tr><tr><td align="left" valign="top" charoff="50"> α,β,γ (°)</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td></tr><tr><td align="left" valign="top" charoff="50">Resolution (Å)</td><td align="center" valign="top" charoff="50">50–1.38 (1.42–1.38)</td><td align="center" valign="top" charoff="50">50–1.60 (1.66–1.60)</td><td align="center" valign="top" charoff="50">50–1.80 (1.86–1.80)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>p.i.m.</sub>(%)<xref ref-type="fn" rid="t1-fn2">**</xref></td><td align="center" valign="top" charoff="50">3.0 (34.4)</td><td align="center" valign="top" charoff="50">2.1 (32.5)</td><td align="center" valign="top" charoff="50">2.6 (47.8)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>I</italic>/<italic>σ</italic></td><td align="center" valign="top" charoff="50">21.5 (2.0)</td><td align="center" valign="top" charoff="50">31.8 (2.0)</td><td align="center" valign="top" charoff="50">28.0 (2.4)</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">99.8 (99.1)</td><td align="center" valign="top" charoff="50">99.6 (98.5)</td><td align="center" valign="top" charoff="50">99.9 (99.7)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">6.9 (5.0)</td><td align="center" valign="top" charoff="50">6.9 (6.2)</td><td align="center" valign="top" charoff="50">6.3 (5.9)</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Refinement</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">25.81–1.38</td><td align="center" valign="top" charoff="50">33.55–1.60</td><td align="center" valign="top" charoff="50">43.52–1.80</td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections</td><td align="center" valign="top" charoff="50">43660</td><td align="center" valign="top" charoff="50">28588</td><td align="center" valign="top" charoff="50">20490</td></tr><tr><td align="left" valign="top" charoff="50"><italic> R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">0.182/0.192</td><td align="center" valign="top" charoff="50">0.181/0.184</td><td align="center" valign="top" charoff="50">0.189/0.209</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">No. atoms</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">1717</td><td align="center" valign="top" charoff="50">1576</td><td align="center" valign="top" charoff="50">1566</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">116</td><td align="center" valign="top" charoff="50">96</td><td align="center" valign="top" charoff="50">96</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">289</td><td align="center" valign="top" charoff="50">258</td><td align="center" valign="top" charoff="50">168</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50"><italic>B</italic>-factors</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">23.8</td><td align="center" valign="top" charoff="50">32.0</td><td align="center" valign="top" charoff="50">37.4</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">22.2</td><td align="center" valign="top" charoff="50">34.6</td><td align="center" valign="top" charoff="50">43.7</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">35.1</td><td align="center" valign="top" charoff="50">46.4</td><td align="center" valign="top" charoff="50">49.1</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">R.m.s. deviations</td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.018</td><td align="center" valign="top" charoff="50">0.017</td><td align="center" valign="top" charoff="50">0.015</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="center" valign="top" charoff="50">1.529</td><td align="center" valign="top" charoff="50">1.651</td><td align="center" valign="top" charoff="50">1.581</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Ramachandran Plot</td></tr><tr><td align="left" valign="top" charoff="50"> Favoured region</td><td align="center" valign="top" charoff="50">98.98%</td><td align="center" valign="top" charoff="50">98.93%</td><td align="center" valign="top" charoff="50">98.96%</td></tr><tr><td align="left" valign="top" charoff="50"> Allowed region</td><td align="center" valign="top" charoff="50">1.02%</td><td align="center" valign="top" charoff="50">1.07%</td><td align="center" valign="top" charoff="50">1.04%</td></tr><tr><td align="left" valign="top" charoff="50"> Outliers</td><td align="center" valign="top" charoff="50">0.00%</td><td align="center" valign="top" charoff="50">0.00%</td><td align="center" valign="top" charoff="50">0.00%</td></tr></tbody></table>
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<text>Structure and PDB ID hNaa60(1-242)/Ac-CoA 5HGZ hNaa60(1-199)/CoA 5HH0 hNaa60(1-199)F34A/CoA 5HH1 Data collection* Space group P212121 P21212 P21212 Cell dimensions a, b, c (Å) 53.3, 57.4, 68.8 67.8, 73.8, 43.2 66.7, 74.0, 43.5 α,β,γ (°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å) 50–1.38 (1.42–1.38) 50–1.60 (1.66–1.60) 50–1.80 (1.86–1.80) Rp.i.m.(%)** 3.0 (34.4) 2.1 (32.5) 2.6 (47.8) I/σ 21.5 (2.0) 31.8 (2.0) 28.0 (2.4) Completeness (%) 99.8 (99.1) 99.6 (98.5) 99.9 (99.7) Redundancy 6.9 (5.0) 6.9 (6.2) 6.3 (5.9) Refinement Resolution (Å) 25.81–1.38 33.55–1.60 43.52–1.80 No. reflections 43660 28588 20490 Rwork/Rfree 0.182/0.192 0.181/0.184 0.189/0.209 No. atoms Protein 1717 1576 1566 Ligand/ion 116 96 96 Water 289 258 168 B-factors Protein 23.8 32.0 37.4 Ligand/ion 22.2 34.6 43.7 Water 35.1 46.4 49.1 R.m.s. deviations Bond lengths (Å) 0.018 0.017 0.015 Bond angles (°) 1.529 1.651 1.581 Ramachandran Plot Favoured region 98.98% 98.93% 98.96% Allowed region 1.02% 1.07% 1.04% Outliers 0.00% 0.00% 0.00% </text>
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<infon key="article-id_doi">10.7554/eLife.18972</infon>
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<infon key="kwd">encapsulin encapsulated ferritin ferritin Rhodospirillum rubrum Other</infon>
<infon key="license">This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.</infon>
<infon key="name_0">surname:He;given-names:Didi</infon>
<infon key="name_1">surname:Hughes;given-names:Sam</infon>
<infon key="name_10">surname:Losick;given-names:Richard</infon>
<infon key="name_11">surname:He;given-names:Didi</infon>
<infon key="name_12">surname:Hughes;given-names:Sam</infon>
<infon key="name_13">surname:Altenbach;given-names:Kirsten</infon>
<infon key="name_14">surname:Clarke;given-names:David J</infon>
<infon key="name_15">surname:Tarrant;given-names:Emma</infon>
<infon key="name_16">surname:Waldron;given-names:Kevin J</infon>
<infon key="name_17">surname:Clarke;given-names:David J</infon>
<infon key="name_18">surname:Marles-Wright;given-names:Jon</infon>
<infon key="name_19">surname:Marles-Wright;given-names:Jon</infon>
<infon key="name_2">surname:Vanden-Hehir;given-names:Sally</infon>
<infon key="name_3">surname:Georgiev;given-names:Atanas</infon>
<infon key="name_4">surname:Altenbach;given-names:Kirsten</infon>
<infon key="name_5">surname:Tarrant;given-names:Emma</infon>
<infon key="name_6">surname:Mackay;given-names:C Logan</infon>
<infon key="name_7">surname:Waldron;given-names:Kevin J</infon>
<infon key="name_8">surname:Clarke;given-names:David J</infon>
<infon key="name_9">surname:Marles-Wright;given-names:Jon</infon>
<infon key="section_type">TITLE</infon>
<infon key="title">Author Keywords Research Organism</infon>
<infon key="type">front</infon>
<infon key="volume">5</infon>
<infon key="year">2016</infon>
<offset>0</offset>
<text>Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments</text>
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<text>Structural characterization</text>
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<infon key="annotator">cleaner0</infon>
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<text>encapsulated</text>
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<text>ferritin</text>
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<text>iron</text>
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<text>nanocompartments</text>
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<text>Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.</text>
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<text>Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells.</text>
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<text>To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.</text>
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<text>Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.</text>
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<text>He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria. The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.</text>
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<text>Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.</text>
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<text>Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.</text>
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<offset>3178</offset>
<text>DOI: http://dx.doi.org/10.7554/eLife.18972.002</text>
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<infon key="section_type">INTRO</infon>
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<text>Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea. They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5. Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits. In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron. The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.</text>
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<text>Encapsulin</text>
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<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
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<text>The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn). Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known. Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage. The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.</text>
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<infon key="updated_at">2023-09-19T16:22:34Z</infon>
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<text>dye-dependent peroxidase</text>
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<infon key="updated_at">2023-09-19T16:22:38Z</infon>
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<text>DyP</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:40:20Z</infon>
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<text>encapsulin-associated ferritins</text>
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<infon key="updated_at">2023-09-19T11:40:27Z</infon>
<infon key="identifier">MESH:</infon>
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<text>EncFtn</text>
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<infon key="updated_at">2023-09-19T11:40:30Z</infon>
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<text>DyP family</text>
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<text>Ferritin</text>
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<text>kingdoms</text>
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<infon key="updated_at">2023-09-19T11:40:47Z</infon>
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<text>ribonucleotide reductase</text>
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<infon key="updated_at">2023-09-19T11:27:29Z</infon>
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<text>iron</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="4552" length="9"/>
<text>classical</text>
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<annotation id="1890">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:41:27Z</infon>
<location offset="4562" length="31"/>
<text>iron storage ferritin nanocages</text>
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<annotation id="119">
<infon key="score">0.80968964</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:40:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4611" length="8"/>
<text>kingdoms</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:56:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4641" length="10"/>
<text>eukaryotes</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:29Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>iron</text>
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<annotation id="122">
<infon key="score">0.9989377</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:41:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4743" length="4"/>
<text>Fe2+</text>
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<infon key="score">0.99878216</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:41:39Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>Fe3+</text>
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<infon key="updated_at">2023-09-19T16:23:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4798" length="12"/>
<text>ferrihydrite</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:12:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4833" length="14"/>
<text>central cavity</text>
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</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>4849</offset>
<text>The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini. This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.</text>
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<infon key="score">0.9172094</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:43:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4853" length="10"/>
<text>encapsulin</text>
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<infon key="score">0.9980197</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:36:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4910" length="11"/>
<text>icosahedral</text>
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<infon key="score">0.9989114</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4922" length="5"/>
<text>shell</text>
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<annotation id="129">
<infon key="score">0.9965553</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4961" length="5"/>
<text>shell</text>
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<infon key="score">0.9985676</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:42:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4989" length="27"/>
<text>short localization sequence</text>
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<annotation id="131">
<infon key="score">0.93535024</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:43:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5018" length="15"/>
<text>Gly-Ser-Leu-Lys</text>
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<annotation id="132">
<infon key="score">0.89328057</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:59:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5064" length="10"/>
<text>This motif</text>
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<annotation id="133">
<infon key="score">0.9987045</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:43:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5078" length="14"/>
<text>well-conserved</text>
</annotation>
<annotation id="134">
<infon key="score">0.90473825</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5201" length="11"/>
<text>encapsulins</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>5217</offset>
<text>A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress. At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins. The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins. Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1). The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins. The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues. The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).</text>
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<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:37:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5239" length="18"/>
<text>Myxococcus xanthus</text>
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<annotation id="136">
<infon key="score">0.99003536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:26Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5258" length="10"/>
<text>encapsulin</text>
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<annotation id="1891">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:31Z</infon>
<location offset="5317" length="6"/>
<text>EncFtn</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<location offset="5349" length="4"/>
<text>iron</text>
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<annotation id="137">
<infon key="score">0.9984309</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5382" length="8"/>
<text>bacteria</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<location offset="5480" length="8"/>
<text>ferritin</text>
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<annotation id="138">
<infon key="score">0.92684335</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5531" length="9"/>
<text>classical</text>
</annotation>
<annotation id="139">
<infon key="score">0.77692854</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5541" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="140">
<infon key="score">0.98316735</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:46Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5550" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="141">
<infon key="score">0.83831835</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5583" length="3"/>
<text>Dps</text>
</annotation>
<annotation id="1910">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:41Z</infon>
<location offset="5588" length="19"/>
<text>DNA-binding protein</text>
</annotation>
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<infon key="score">0.99889994</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5694" length="4"/>
<text>iron</text>
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<infon key="score">0.9989184</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5710" length="9"/>
<text>ferritins</text>
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<annotation id="1854">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:48Z</infon>
<location offset="5746" length="6"/>
<text>EncFtn</text>
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<annotation id="144">
<infon key="score">0.9775484</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:45:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5767" length="11"/>
<text>Glu-X-X-His</text>
</annotation>
<annotation id="145">
<infon key="score">0.8720136</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:45:46Z</infon>
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<location offset="5779" length="24"/>
<text>metal coordination sites</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<location offset="5838" length="8"/>
<text>ferritin</text>
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<infon key="score">0.9923527</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5864" length="30"/>
<text>Secondary structure prediction</text>
</annotation>
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<infon key="score">0.9919742</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5910" length="23"/>
<text>major α-helical regions</text>
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<annotation id="1837">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<location offset="5997" length="8"/>
<text>ferritin</text>
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<annotation id="148">
<infon key="score">0.81719357</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:48:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6035" length="15"/>
<text>major α-helices</text>
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<annotation id="1838">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<location offset="6085" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1855">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:48:47Z</infon>
<location offset="6119" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="149">
<infon key="score">0.646938</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:26Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6160" length="10"/>
<text>encapsulin</text>
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<annotation id="150">
<infon key="score">0.8150764</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="6171" length="16"/>
<text>nanocompartments</text>
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<annotation id="1839">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<location offset="6257" length="8"/>
<text>ferritin</text>
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<infon key="score">0.99869084</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6287" length="21"/>
<text>Rhodospirillum rubrum</text>
</annotation>
<annotation id="152">
<infon key="score">0.8986397</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6309" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1915">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:49:38Z</infon>
<location offset="6325" length="9"/>
<text>Rru_A0973</text>
</annotation>
<annotation id="153">
<infon key="score">0.9987142</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6382" length="10"/>
<text>M. xanthus</text>
</annotation>
<annotation id="1916">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:49:56Z</infon>
<location offset="6394" length="9"/>
<text>MXAN_4464</text>
</annotation>
<annotation id="154">
<infon key="score">0.9986573</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6419" length="11"/>
<text>T. maritima</text>
</annotation>
<annotation id="1917">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:14Z</infon>
<location offset="6432" length="10"/>
<text>Tmari_0787</text>
</annotation>
<annotation id="155">
<infon key="score">0.99866563</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6462" length="11"/>
<text>P. furiosus</text>
</annotation>
<annotation id="156">
<infon key="score">0.5972664</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:55Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="6475" length="6"/>
<text>PF1192</text>
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<annotation id="1918">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:51:21Z</infon>
<location offset="6499" length="4"/>
<text>GXXH</text>
</annotation>
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<infon key="score">0.9986265</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:51:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6515" length="18"/>
<text>strictly conserved</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>6594</offset>
<text>Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment. We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli. Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn. The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins. We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface. We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry. Biochemical assays show that EncFtn is active as a ferroxidase enzyme. Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity. We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.</text>
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<infon key="score">0.98747754</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:51:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6618" length="9"/>
<text>structure</text>
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<infon key="score">0.99886274</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6648" length="6"/>
<text>EncFtn</text>
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<infon key="score">0.99871385</infon>
<infon key="type">chemical</infon>
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<text>Full-frame transmission electron micrographs of R. rubrum nanocompartments.</text>
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<text>Full-frame transmission electron micrographs</text>
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<text>(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments. All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters. A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).</text>
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<text>Negative stain TEM</text>
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<text>image</text>
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<text>R. rubrum</text>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<text>encapsulin</text>
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<text>EncFtn-Enc</text>
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<infon key="updated_at">2023-09-19T11:47:29Z</infon>
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<text>nanocompartments</text>
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<text>Histogram</text>
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<infon key="updated_at">2023-09-19T17:56:57Z</infon>
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<text>nanocompartment</text>
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<infon key="updated_at">2023-09-19T12:00:14Z</infon>
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<location offset="8560" length="40"/>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<text>encapsulin</text>
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<infon key="updated_at">2023-09-19T11:30:37Z</infon>
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<text>encapsulin</text>
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<infon key="updated_at">2023-09-19T12:00:07Z</infon>
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<text>EncFtn-Enc</text>
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<infon key="file">elife-18972-fig1-figsupp1.jpg</infon>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.004</text>
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<passage>
<infon key="file">elife-18972-fig1.jpg</infon>
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<infon key="type">fig_title_caption</infon>
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<text>Purification of recombinant R. rubrum encapsulin nanocompartments.</text>
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<text>R. rubrum</text>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
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<text>(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium. Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows. (B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments. Samples were imaged at 143,000 x magnification, with scale bar shown as 25 nm. Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.</text>
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<infon key="updated_at">2023-09-19T12:00:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8963" length="23"/>
<text>Recombinantly expressed</text>
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<infon key="score">0.9987801</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8987" length="10"/>
<text>encapsulin</text>
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<annotation id="238">
<infon key="score">0.99761957</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:11:34Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8999" length="3"/>
<text>Enc</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:31Z</infon>
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<infon key="updated_at">2023-09-19T12:00:07Z</infon>
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<text>EncFtn-Enc</text>
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<infon key="updated_at">2023-09-19T12:00:57Z</infon>
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<text>sucrose gradient ultracentrifugation</text>
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<location offset="9091" length="7"/>
<text>E. coli</text>
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<annotation id="1963">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:16:44Z</infon>
<location offset="9118" length="5"/>
<text>SeMet</text>
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<text>SDS-PAGE</text>
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<infon key="updated_at">2023-09-19T11:59:52Z</infon>
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<text>Negative stain TEM</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<text>encapsulin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:07Z</infon>
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<text>EncFtn-Enc</text>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.003</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>9578</offset>
<text>We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A). TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin. We were not able to resolve any higher-order structures of EncFtn by TEM. Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).</text>
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<text>R. rubrum</text>
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<infon key="annotator">cleaner0</infon>
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<infon key="updated_at">2023-09-19T11:56:09Z</infon>
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<infon key="updated_at">2023-09-19T18:19:49Z</infon>
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<text>encapsulin</text>
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<infon key="identifier">GENE:</infon>
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<infon key="updated_at">2023-09-19T11:30:37Z</infon>
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<infon key="identifier">GENE:</infon>
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<infon key="updated_at">2023-09-19T13:14:57Z</infon>
<location offset="9710" length="9"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:53Z</infon>
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<text>sucrose gradient ultra-centrifugation</text>
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<infon key="updated_at">2023-09-19T11:52:14Z</infon>
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<text>TEM</text>
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<infon key="updated_at">2023-09-19T13:15:43Z</infon>
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<text>expressed in isolation</text>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<text>encapsulin</text>
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<infon key="updated_at">2023-09-19T16:15:20Z</infon>
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<text>empty</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:15:29Z</infon>
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<text>compartments</text>
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<infon key="updated_at">2023-09-19T11:50:39Z</infon>
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<text>T. maritima</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
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<infon key="updated_at">2023-09-19T11:30:37Z</infon>
<infon key="identifier">PR:</infon>
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<text>EncFtn</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:14Z</infon>
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<text>TEM</text>
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<text>encapsulin</text>
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<text>Purification of recombinant R. rubrum EncFtnsH.</text>
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<text>R. rubrum</text>
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<infon key="updated_at">2023-09-19T13:12:40Z</infon>
<location offset="10542" length="8"/>
<text>EncFtnsH</text>
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<text>(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare). Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment. The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards. Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis. (B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting. The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions. (C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).</text>
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<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T13:17:56Z</infon>
<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-19T13:12:51Z</infon>
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<infon key="updated_at">2023-09-19T13:12:58Z</infon>
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<infon key="updated_at">2023-09-19T13:19:33Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-19T13:13:06Z</infon>
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<text>ICP-MS</text>
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<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-19T13:19:19Z</infon>
<location offset="10985" length="16"/>
<text>molecular weight</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-19T13:13:55Z</infon>
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<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T13:13:39Z</infon>
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<text>monomer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11246" length="4"/>
<text>iron</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
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<text>ICP-MS</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:18Z</infon>
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<text>gel filtration</text>
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<infon key="updated_at">2023-09-19T13:13:25Z</infon>
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<text>SDS-PAGE</text>
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<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11464" length="8"/>
<text>EncFtnsH</text>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:02Z</infon>
<infon key="identifier">MESH:</infon>
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<text>MALDI peptide mass fingerprinting</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:38Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>monomer</text>
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<annotation id="294">
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<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:15:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11628" length="5"/>
<text>dimer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11637" length="8"/>
<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T18:16:05Z</infon>
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<text>dimer</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>decamer</text>
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<infon key="updated_at">2023-09-19T13:13:32Z</infon>
<infon key="identifier">MESH:</infon>
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<text>SEC-MALLS</text>
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<infon key="updated_at">2023-09-19T13:13:55Z</infon>
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<text>EncFtnsH</text>
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<annotation id="300">
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11744" length="7"/>
<text>decamer</text>
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<infon key="updated_at">2023-09-19T13:13:39Z</infon>
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<text>monomer</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<text>decamer</text>
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<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>monomer</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>monomer</text>
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<infon key="file">elife-18972-fig2.jpg</infon>
<infon key="id">fig2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>11944</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.005</text>
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<passage>
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<offset>11991</offset>
<text>Determination of the Fe/EncFtnsH protein ratio by ICP-MS. EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2. Fractions from SEC were collected, acidified and analysed by ICP-MS. EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer. Samples where the element was undetectable are labelled with n.d. These data were collected from EncFtnsH fractions from a single gel-filtration run.</text>
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<infon key="updated_at">2023-09-19T16:23:07Z</infon>
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<text>Fe</text>
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<infon key="updated_at">2023-09-19T13:13:55Z</infon>
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<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T13:16:43Z</infon>
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<text>SeMet</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:48Z</infon>
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<text>E. coli B834(DE3)</text>
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<infon key="updated_at">2023-09-19T13:16:44Z</infon>
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<text>SeMet</text>
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<infon key="updated_at">2023-09-19T13:17:55Z</infon>
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<infon key="updated_at">2023-09-19T13:18:13Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
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<text>ICP-MS</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12239" length="8"/>
<text>EncFtnsH</text>
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<text>Peak EncFtnsHretention volume (ml) Element concentration (µM) Derived EncFtnsHconcentration (µM) Derived Fe/ EncFtnsH monomer Ca Fe Zn Se Decamer 66.5 n.d. 6.7 n.d. 24.6 12.3 0.5 68.3 n.d. 28.4 n.d 124.5 62.3 0.5 70.1 2.9 93.7 2.4 301.7 150.9 0.6 71.9 6.9 120.6 3.7 379.8 189.9 0.6 73.7 1.9 64.4 0.8 240.6 120.3 0.5 75.5 0.9 21.1 n.d. 101.7 50.8 0.4 77.3 n.d. 6.2 n.d. 42.6 21.3 0.3 79.1 0.1 2.4 n.d. 26.5 13.3 0.2 80.9 1.0 1.5 n.d. 22.3 11.2 0.1 82.7 n.d. 0.2 n.d. 29.2 14.6 n.d Monomer 84.5 n.d. 0.1 n.d. 34.9 17.5 n.d 86.3 n.d. n.d n.d. 28.9 14.4 n.d 88.1 n.d. n.d. n.d. 17.4 8.7 n.d. 89.9 n.d. n.d. n.d. 5.5 2.8 n.d. 91.7 n.d. n.d. n.d. 0.1 0.07 0.2 </text>
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<text>Estimates of EncFtnsH molecular weight from SEC-MALLS analysis. EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography. Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C). The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions. The molecular weights are quoted to the nearest kDa due to the resolution limit of the instrument. The proteins analyzed by SEC-MALLS came from single protein preparation.</text>
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<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Molecular Weight (kDa)</th><th rowspan="1" colspan="1">Decamer peak</th><th rowspan="1" colspan="1">Monomer peak</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">Theoretical</td><td rowspan="1" colspan="1">133</td><td rowspan="1" colspan="1">13</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-decamer fractions</td><td rowspan="1" colspan="1">132</td><td rowspan="1" colspan="1">15</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">126</td><td rowspan="1" colspan="1">13</td></tr></tbody></table>
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<text>Molecular Weight (kDa) Decamer peak Monomer peak Theoretical 133 13 EncFtnsH-decamer fractions 132 15 EncFtnsH-monomer fractions 126 13 </text>
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<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14067" length="7"/>
<text>Decamer</text>
</annotation>
<annotation id="341">
<infon key="score">0.99871075</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14080" length="7"/>
<text>Monomer</text>
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<annotation id="1938">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<location offset="14116" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="342">
<infon key="score">0.99848974</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14125" length="7"/>
<text>decamer</text>
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<annotation id="343">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14152" length="8"/>
<text>EncFtnsH</text>
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<annotation id="344">
<infon key="score">0.99874073</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14161" length="7"/>
<text>monomer</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>14189</offset>
<text>We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH). In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A). SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B). MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding. In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1). In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins. Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:56:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14213" length="9"/>
<text>R. rubrum</text>
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<infon key="score">0.9992718</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14223" length="6"/>
<text>EncFtn</text>
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<annotation id="347">
<infon key="score">0.99910957</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14242" length="11"/>
<text>full-length</text>
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<annotation id="348">
<infon key="score">0.8145341</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14264" length="15"/>
<text>140 amino acids</text>
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<annotation id="349">
<infon key="score">0.9933616</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14287" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="350">
<infon key="score">0.9985709</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14308" length="20"/>
<text>hexahistidine-tagged</text>
</annotation>
<annotation id="351">
<infon key="score">0.997155</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14350" length="4"/>
<text>1–96</text>
</annotation>
<annotation id="352">
<infon key="score">0.9950584</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14376" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2117">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:22Z</infon>
<location offset="14405" length="15"/>
<text>elution profile</text>
</annotation>
<annotation id="353">
<infon key="score">0.9990057</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:12:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14426" length="29"/>
<text>size-exclusion chromatography</text>
</annotation>
<annotation id="354">
<infon key="score">0.99856037</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:18:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14457" length="3"/>
<text>SEC</text>
</annotation>
<annotation id="1986">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:26:00Z</infon>
<location offset="14476" length="5"/>
<text>peaks</text>
</annotation>
<annotation id="355">
<infon key="score">0.9989311</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14495" length="8"/>
<text>SDS-PAGE</text>
</annotation>
<annotation id="1987">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:26:00Z</infon>
<location offset="14537" length="5"/>
<text>peaks</text>
</annotation>
<annotation id="1973">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:19:19Z</infon>
<location offset="14564" length="16"/>
<text>molecular weight</text>
</annotation>
<annotation id="1974">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:19:19Z</infon>
<location offset="14669" length="16"/>
<text>molecular weight</text>
</annotation>
<annotation id="356">
<infon key="score">0.9988757</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:16:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14711" length="9"/>
<text>monomeric</text>
</annotation>
<annotation id="357">
<infon key="score">0.99877673</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14749" length="33"/>
<text>MALDI peptide mass fingerprinting</text>
</annotation>
<annotation id="358">
<infon key="score">0.99930024</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14832" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="359">
<infon key="score">0.9988841</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14840" length="44"/>
<text>Inductively coupled plasma mass spectrometry</text>
</annotation>
<annotation id="360">
<infon key="score">0.99878293</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14886" length="6"/>
<text>ICP-MS</text>
</annotation>
<annotation id="361">
<infon key="score">0.9979761</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:18:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14910" length="3"/>
<text>SEC</text>
</annotation>
<annotation id="362">
<infon key="score">0.9991605</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14946" length="4"/>
<text>iron</text>
</annotation>
<annotation id="363">
<infon key="score">0.9989423</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14987" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="364">
<infon key="score">0.99928397</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15054" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="365">
<infon key="score">0.9990219</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15096" length="4"/>
<text>iron</text>
</annotation>
<annotation id="366">
<infon key="score">0.99906236</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15136" length="4"/>
<text>iron</text>
</annotation>
<annotation id="367">
<infon key="score">0.99860877</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15170" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="368">
<infon key="score">0.9987936</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15194" length="6"/>
<text>ICP-MS</text>
</annotation>
<annotation id="369">
<infon key="score">0.9991273</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15234" length="16"/>
<text>selenomethionine</text>
</annotation>
<annotation id="370">
<infon key="score">0.9992005</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:16:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15252" length="5"/>
<text>SeMet</text>
</annotation>
<annotation id="371">
<infon key="score">0.9992411</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15276" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1893">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="15390" length="9"/>
<text>classical</text>
</annotation>
<annotation id="372">
<infon key="score">0.99904937</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15400" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="373">
<infon key="score">0.99899334</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:12:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15411" length="29"/>
<text>Size-exclusion chromatography</text>
</annotation>
<annotation id="374">
<infon key="score">0.9956793</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15446" length="34"/>
<text>multi-angle laser light scattering</text>
</annotation>
<annotation id="375">
<infon key="score">0.99888945</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15482" length="9"/>
<text>SEC-MALLS</text>
</annotation>
<annotation id="376">
<infon key="score">0.9988186</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15586" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1975">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:19:19Z</infon>
<location offset="15607" length="16"/>
<text>molecular weight</text>
</annotation>
<annotation id="377">
<infon key="score">0.998869</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15635" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1976">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:19:19Z</infon>
<location offset="15655" length="16"/>
<text>molecular weight</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp1.jpg</infon>
<infon key="id">fig3s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>15702</offset>
<text>Effect of metal ions on the oligomeric state of EncFtnsH in solution.</text>
<annotation id="378">
<infon key="score">0.998884</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15750" length="8"/>
<text>EncFtnsH</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp1.jpg</infon>
<infon key="id">fig3s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>15773</offset>
<text>(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column. Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.</text>
<annotation id="379">
<infon key="score">0.6916507</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15779" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="380">
<infon key="score">0.99887174</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15788" length="7"/>
<text>monomer</text>
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<annotation id="381">
<infon key="score">0.96875036</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15800" length="9"/>
<text>incubated</text>
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<annotation id="382">
<infon key="score">0.9985541</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15881" length="25"/>
<text>analytical gel-filtration</text>
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<annotation id="1978">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:21:43Z</infon>
<location offset="15946" length="4"/>
<text>Co2+</text>
</annotation>
<annotation id="383">
<infon key="score">0.9992772</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:21:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15955" length="4"/>
<text>Zn2+</text>
</annotation>
<annotation id="384">
<infon key="score">0.99869174</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15989" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="385">
<infon key="score">0.9992417</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16007" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1998">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:28:59Z</infon>
<location offset="16023" length="4"/>
<text>Mn2+</text>
</annotation>
<annotation id="1979">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:10Z</infon>
<location offset="16029" length="4"/>
<text>Mg2+</text>
</annotation>
<annotation id="386">
<infon key="score">0.99926716</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:13Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16038" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="387">
<infon key="score">0.99925584</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16095" length="8"/>
<text>EncFtnsH</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp1.jpg</infon>
<infon key="id">fig3s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16105</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.009</text>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp2.jpg</infon>
<infon key="id">fig3s2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>16152</offset>
<text>PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.</text>
<annotation id="388">
<infon key="score">0.99873024</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16152" length="4"/>
<text>PAGE</text>
</annotation>
<annotation id="389">
<infon key="score">0.99764544</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16221" length="8"/>
<text>EncFtnsH</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp2.jpg</infon>
<infon key="id">fig3s2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16231</offset>
<text>50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE. (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.</text>
<annotation id="390">
<infon key="score">0.99864906</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16237" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="391">
<infon key="score">0.99867463</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16246" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="392">
<infon key="score">0.99853146</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16257" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="393">
<infon key="score">0.998766</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16322" length="5"/>
<text>Fe2+,</text>
</annotation>
<annotation id="394">
<infon key="score">0.99845093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16328" length="5"/>
<text>Co2+,</text>
</annotation>
<annotation id="395">
<infon key="score">0.9981501</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16334" length="5"/>
<text>Zn2+,</text>
</annotation>
<annotation id="396">
<infon key="score">0.99844736</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16340" length="5"/>
<text>Mn2+,</text>
</annotation>
<annotation id="397">
<infon key="score">0.9960065</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16346" length="5"/>
<text>Ca2+,</text>
</annotation>
<annotation id="398">
<infon key="score">0.9992015</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16352" length="4"/>
<text>Mg2+</text>
</annotation>
<annotation id="399">
<infon key="score">0.9966721</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:22:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16361" length="5"/>
<text>Fe3+,</text>
</annotation>
<annotation id="400">
<infon key="score">0.99871135</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16390" length="11"/>
<text>Native PAGE</text>
</annotation>
<annotation id="401">
<infon key="score">0.9988149</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16412" length="8"/>
<text>SDS-PAGE</text>
</annotation>
<annotation id="402">
<infon key="score">0.9985031</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16430" length="11"/>
<text>Native PAGE</text>
</annotation>
<annotation id="403">
<infon key="score">0.99836725</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16454" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="404">
<infon key="score">0.99835724</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16463" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="405">
<infon key="score">0.99853516</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16525" length="11"/>
<text>Native PAGE</text>
</annotation>
<annotation id="406">
<infon key="score">0.9980385</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16549" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="407">
<infon key="score">0.9972319</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16558" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="408">
<infon key="score">0.9987367</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16620" length="8"/>
<text>SDS-PAGE</text>
</annotation>
<annotation id="409">
<infon key="score">0.9985697</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16657" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="410">
<infon key="score">0.99824524</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16666" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="411">
<infon key="score">0.9987531</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16713" length="8"/>
<text>SDS-PAGE</text>
</annotation>
<annotation id="412">
<infon key="score">0.9983241</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16750" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="413">
<infon key="score">0.997407</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16759" length="7"/>
<text>decamer</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3-figsupp2.jpg</infon>
<infon key="id">fig3s2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16802</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.010</text>
</passage>
<passage>
<infon key="file">elife-18972-fig3.jpg</infon>
<infon key="id">fig3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>16849</offset>
<text>Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.</text>
<annotation id="414">
<infon key="score">0.99921703</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:23:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16859" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="415">
<infon key="score">0.99787915</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16917" length="8"/>
<text>EncFtnsH</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig3.jpg</infon>
<infon key="id">fig3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16939</offset>
<text>(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+). A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH
in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare). Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml. Monomeric and decameric samples of EncFtnsH are shown as controls. Peaks around 0.8 ml were seen as protein aggregation. (C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization. The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.</text>
<annotation id="416">
<infon key="score">0.99880147</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16955" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1933">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:20Z</infon>
<location offset="16980" length="14"/>
<text>Gel filtration</text>
</annotation>
<annotation id="1994">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:26:29Z</infon>
<location offset="17028" length="17"/>
<text>E. coli BL21(DE3)</text>
</annotation>
<annotation id="2002">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="17055" length="2"/>
<text>MM</text>
</annotation>
<annotation id="2003">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="17064" length="2"/>
<text>MM</text>
</annotation>
<annotation id="417">
<infon key="score">0.99887115</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17090" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
</annotation>
<annotation id="2004">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="17106" length="2"/>
<text>MM</text>
</annotation>
<annotation id="2017">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:31:36Z</infon>
<location offset="17109" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="418">
<infon key="score">0.99884593</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17139" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="2005">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="17211" length="2"/>
<text>MM</text>
</annotation>
<annotation id="2018">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:31:55Z</infon>
<location offset="17214" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="2019">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:32:23Z</infon>
<location offset="17231" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2006">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="17240" length="2"/>
<text>MM</text>
</annotation>
<annotation id="419">
<infon key="score">0.9989965</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17260" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="420">
<infon key="score">0.99917525</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17300" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="421">
<infon key="score">0.99196833</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17322" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="422">
<infon key="score">0.9988833</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17331" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="423">
<infon key="score">0.99834794</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17382" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="424">
<infon key="score">0.99785185</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17416" length="25"/>
<text>analytical gel-filtration</text>
</annotation>
<annotation id="425">
<infon key="score">0.9989563</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:40Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17502" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="426">
<infon key="score">0.9988194</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17545" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="427">
<infon key="score">0.998934</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:16:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17599" length="9"/>
<text>Monomeric</text>
</annotation>
<annotation id="428">
<infon key="score">0.9987066</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17613" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="429">
<infon key="score">0.9989278</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17634" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1983">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:25:59Z</infon>
<location offset="17666" length="5"/>
<text>Peaks</text>
</annotation>
<annotation id="430">
<infon key="score">0.9983282</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:27Z</infon>
<infon key="identifier">MESH:</infon>
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<text>Analytical gel filtration</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17753" length="6"/>
<text>EncFtn</text>
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<infon key="annotator">cleaner0</infon>
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<text>monomer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:16:28Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>dimer</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>decamer</text>
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<infon key="id">fig3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>18097</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.008</text>
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<passage>
<infon key="file">tbl3.xml</infon>
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<infon key="section_type">TABLE</infon>
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<offset>18144</offset>
<text>Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.</text>
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<text>Gel-filtration</text>
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<infon key="updated_at">2023-09-19T13:25:42Z</infon>
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<text>peak area ratios</text>
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<infon key="updated_at">2023-09-19T13:13:55Z</infon>
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<location offset="18180" length="8"/>
<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<text>decamer</text>
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<infon key="score">0.99890876</infon>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<location offset="18201" length="7"/>
<text>monomer</text>
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<location offset="18246" length="8"/>
<text>EncFtnsH</text>
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<infon key="identifier">MESH:</infon>
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<location offset="18337" length="2"/>
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<text>Fe2+</text>
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<infon key="updated_at">2023-09-19T18:20:21Z</infon>
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<location offset="18362" length="29"/>
<text>gel-filtration chromatography</text>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18444" length="7"/>
<text>Monomer</text>
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<text>EncFtnsH</text>
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<text>EncFtnsH</text>
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<text>monomer</text>
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<infon key="updated_at">2023-09-19T13:33:20Z</infon>
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<text>analytical gel filtration chromatography</text>
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<text>AGF</text>
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<annotation id="1988">
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<infon key="updated_at">2023-09-19T13:26:00Z</infon>
<location offset="18845" length="5"/>
<text>peaks</text>
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<text>peak ratios</text>
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<text>EncFtnsH</text>
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<location offset="19000" length="11"/>
<text>presence of</text>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<text>monomer</text>
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<infon key="score">0.9988072</infon>
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<text>decamer</text>
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<infon key="score">0.9988734</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<text>decamer</text>
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<infon key="score">0.9987771</infon>
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<location offset="19260" length="7"/>
<text>decamer</text>
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<infon key="score">0.9985372</infon>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<location offset="19269" length="7"/>
<text>monomer</text>
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<infon key="score">0.9982883</infon>
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<infon key="updated_at">2023-09-19T14:18:27Z</infon>
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<text>presence of</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:27:32Z</infon>
<location offset="19307" length="4"/>
<text>Fe2+</text>
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<annotation id="1997">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:27:47Z</infon>
<location offset="19313" length="4"/>
<text>Co2+</text>
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<infon key="score">0.99925315</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:27:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19323" length="4"/>
<text>Zn2+</text>
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<infon key="score">0.9991636</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
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<location offset="19394" length="8"/>
<text>EncFtnsH</text>
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<infon key="score">0.99849516</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
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<text>analytical gel filtration</text>
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</passage>
<passage>
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<infon key="id">tbl3</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>19662</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.011</text>
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<passage>
<infon key="file">tbl3.xml</infon>
<infon key="id">tbl3</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table</infon>
<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Method</th><th rowspan="1" colspan="1">Sample</th><th rowspan="1" colspan="1">Monomer area</th><th rowspan="1" colspan="1">Decamer area</th><th rowspan="1" colspan="1">Decamer/Monomer</th></tr></thead><tbody><tr><td rowspan="2" colspan="1">Gel filtration Superdex 200 chromatography</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-MM</td><td rowspan="1" colspan="1">64.3</td><td rowspan="1" colspan="1">583.6</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-MM+Fe<sup>2+</sup></td><td rowspan="1" colspan="1">1938.4</td><td rowspan="1" colspan="1">426.4</td><td rowspan="1" colspan="1">4.5</td></tr><tr><td rowspan="4" colspan="1">Analytical Gel filtration Day1</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-decamer fractions</td><td rowspan="1" colspan="1">20.2</td><td rowspan="1" colspan="1">1.8</td><td rowspan="1" colspan="1">11.2</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">2.9</td><td rowspan="1" colspan="1">21.9</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">Fe(NH<sub>4)2</sub>(SO<sub>4)2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">11.0</td><td rowspan="1" colspan="1">13.0</td><td rowspan="1" colspan="1">0.8</td></tr><tr><td rowspan="1" colspan="1">FeSO<sub>4</sub>-HCl/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">11.3</td><td rowspan="1" colspan="1">11.4</td><td rowspan="1" colspan="1">1.0</td></tr><tr><td rowspan="5" colspan="1">Analytical Gel filtration Day2</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">8.3</td><td rowspan="1" colspan="1">22.8</td><td rowspan="1" colspan="1">0.4</td></tr><tr><td rowspan="1" colspan="1">CoCl<sub>2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">17.7</td><td rowspan="1" colspan="1">14.5</td><td rowspan="1" colspan="1">1.2</td></tr><tr><td rowspan="1" colspan="1">MnCl<sub>2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">3.1</td><td rowspan="1" colspan="1">30.5</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">ZnSO<sub>4</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">20.4</td><td rowspan="1" colspan="1">9.0</td><td rowspan="1" colspan="1">2.3</td></tr><tr><td rowspan="1" colspan="1">FeCl<sub>3</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">3.9</td><td rowspan="1" colspan="1">28.6</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="3" colspan="1">Analytical Gel filtration Day3</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">6.3</td><td rowspan="1" colspan="1">23.4</td><td rowspan="1" colspan="1">0.3</td></tr><tr><td rowspan="1" colspan="1">MgSO<sub>4</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">5.8</td><td rowspan="1" colspan="1">30.2</td><td rowspan="1" colspan="1">0.2</td></tr><tr><td rowspan="1" colspan="1">Ca acetate/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">5.6</td><td rowspan="1" colspan="1">25.2</td><td rowspan="1" colspan="1">0.2</td></tr></tbody></table>
</infon>
<offset>19709</offset>
<text>Method Sample Monomer area Decamer area Decamer/Monomer Gel filtration Superdex 200 chromatography EncFtnsH-MM 64.3 583.6 0.1 EncFtnsH-MM+Fe2+ 1938.4 426.4 4.5 Analytical Gel filtration Day1 EncFtnsH-decamer fractions 20.2 1.8 11.2 EncFtnsH-monomer fractions 2.9 21.9 0.1 Fe(NH4)2(SO4)2/EncFtnsH-monomer 11.0 13.0 0.8 FeSO4-HCl/EncFtnsH-monomer 11.3 11.4 1.0 Analytical Gel filtration Day2 EncFtnsH-monomer fractions 8.3 22.8 0.4 CoCl2/EncFtnsH-monomer 17.7 14.5 1.2 MnCl2/EncFtnsH-monomer 3.1 30.5 0.1 ZnSO4/EncFtnsH-monomer 20.4 9.0 2.3 FeCl3/EncFtnsH-monomer 3.9 28.6 0.1 Analytical Gel filtration Day3 EncFtnsH-monomer fractions 6.3 23.4 0.3 MgSO4/EncFtnsH-monomer 5.8 30.2 0.2 Ca acetate/EncFtnsH-monomer 5.6 25.2 0.2 </text>
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<text>Monomer</text>
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<infon key="annotator">cleaner0</infon>
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<text>Decamer</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<text>Decamer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:20Z</infon>
<location offset="19767" length="14"/>
<text>Gel filtration</text>
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<annotation id="1939">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="19810" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2011">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="19819" length="2"/>
<text>MM</text>
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<annotation id="1940">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="19839" length="8"/>
<text>EncFtnsH</text>
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<annotation id="2012">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="19848" length="2"/>
<text>MM</text>
</annotation>
<annotation id="463">
<infon key="score">0.9977776</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19851" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1980">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:28Z</infon>
<location offset="19875" length="25"/>
<text>Analytical Gel filtration</text>
</annotation>
<annotation id="1941">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="19906" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="464">
<infon key="score">0.9542431</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19915" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1942">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="19949" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="465">
<infon key="score">0.9826019</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19958" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="466">
<infon key="score">0.9944018</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19991" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
</annotation>
<annotation id="1943">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20006" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="467">
<infon key="score">0.9878542</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20015" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="468">
<infon key="score">0.996411</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20039" length="9"/>
<text>FeSO4-HCl</text>
</annotation>
<annotation id="1944">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20049" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="469">
<infon key="score">0.9771757</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20058" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1981">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:28Z</infon>
<location offset="20082" length="25"/>
<text>Analytical Gel filtration</text>
</annotation>
<annotation id="1945">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20113" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="470">
<infon key="score">0.9748615</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20122" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="471">
<infon key="score">0.99800307</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20155" length="5"/>
<text>CoCl2</text>
</annotation>
<annotation id="1946">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20161" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="472">
<infon key="score">0.9448928</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20170" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="473">
<infon key="score">0.9977731</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20194" length="5"/>
<text>MnCl2</text>
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<annotation id="1947">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20200" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="474">
<infon key="score">0.91481704</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20209" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="475">
<infon key="score">0.9984397</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:23:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20232" length="5"/>
<text>ZnSO4</text>
</annotation>
<annotation id="1948">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20238" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="476">
<infon key="score">0.892505</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20247" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="477">
<infon key="score">0.99839336</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:24:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20270" length="5"/>
<text>FeCl3</text>
</annotation>
<annotation id="1949">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20276" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="478">
<infon key="score">0.9367046</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20285" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1982">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:24:28Z</infon>
<location offset="20308" length="25"/>
<text>Analytical Gel filtration</text>
</annotation>
<annotation id="1950">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20339" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="479">
<infon key="score">0.9838058</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20348" length="7"/>
<text>monomer</text>
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<annotation id="480">
<infon key="score">0.9920035</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:24:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20381" length="5"/>
<text>MgSO4</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20387" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="481">
<infon key="score">0.8946056</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20396" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="2021">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:34:49Z</infon>
<location offset="20419" length="10"/>
<text>Ca acetate</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<location offset="20430" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="482">
<infon key="score">0.9385806</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20439" length="7"/>
<text>monomer</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>20464</offset>
<text>We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2. The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3). To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE. Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2). While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron. To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C). At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.</text>
<annotation id="483">
<infon key="score">0.9993337</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20476" length="8"/>
<text>EncFtnsH</text>
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<infon key="score">0.9983441</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:56:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20490" length="7"/>
<text>E. coli</text>
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<annotation id="2013">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="20507" length="2"/>
<text>MM</text>
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<infon key="score">0.9990759</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20547" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
</annotation>
<annotation id="486">
<infon key="score">0.9987035</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20567" length="7"/>
<text>decamer</text>
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<annotation id="487">
<infon key="score">0.9987834</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20578" length="7"/>
<text>monomer</text>
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<annotation id="488">
<infon key="score">0.98928785</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20635" length="4"/>
<text>iron</text>
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<annotation id="489">
<infon key="score">0.8405842</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20688" length="9"/>
<text>iron-free</text>
</annotation>
<annotation id="490">
<infon key="score">0.9988404</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20730" length="4"/>
<text>iron</text>
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<infon key="score">0.9993291</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20766" length="8"/>
<text>EncFtnsH</text>
</annotation>
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<infon key="score">0.99936074</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20852" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="493">
<infon key="score">0.792685</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20874" length="9"/>
<text>incubated</text>
</annotation>
<annotation id="494">
<infon key="score">0.9985454</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20948" length="14"/>
<text>analytical SEC</text>
</annotation>
<annotation id="495">
<infon key="score">0.9989158</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:36:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20967" length="19"/>
<text>non-denaturing PAGE</text>
</annotation>
<annotation id="496">
<infon key="score">0.99724555</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21015" length="5"/>
<text>Fe2+,</text>
</annotation>
<annotation id="497">
<infon key="score">0.9993093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21021" length="4"/>
<text>Zn2+</text>
</annotation>
<annotation id="498">
<infon key="score">0.9993104</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:28Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21030" length="4"/>
<text>Co2+</text>
</annotation>
<annotation id="499">
<infon key="score">0.9988109</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21087" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="500">
<infon key="score">0.999256</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21146" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="501">
<infon key="score">0.99936944</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21182" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="502">
<infon key="score">0.99924195</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21192" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="503">
<infon key="score">0.9993135</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:35:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21212" length="5"/>
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<infon key="updated_at">2023-09-19T11:32:18Z</infon>
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<infon key="updated_at">2023-09-19T18:16:34Z</infon>
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<location offset="21689" length="8"/>
<text>dimerize</text>
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<text>Crystal structure of EncFtnsH</text>
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<location offset="21746" length="8"/>
<text>EncFtnsH</text>
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<infon key="type">fig_title_caption</infon>
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<text>Electrostatic surface of EncFtnsH.</text>
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<text>The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL. Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis. (B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.</text>
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<text>EncFtnsH</text>
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<text>EncFtnsH</text>
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<text>central cavity</text>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>22212</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.013</text>
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<passage>
<infon key="file">elife-18972-fig4.jpg</infon>
<infon key="id">fig4</infon>
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<infon key="type">fig_title_caption</infon>
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<text>Crystal structure of EncFtnsH.</text>
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<location offset="22259" length="17"/>
<text>Crystal structure</text>
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<infon key="updated_at">2023-09-19T13:13:56Z</infon>
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<text>EncFtnsH</text>
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<passage>
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<infon key="id">fig4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>22290</offset>
<text>(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres. Alternating subunits are colored blue and green for clarity. The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity. Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.</text>
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<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22318" length="8"/>
<text>EncFtnsH</text>
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<annotation id="520">
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<infon key="updated_at">2023-09-19T13:36:49Z</infon>
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<location offset="22377" length="9"/>
<text>α-helices</text>
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<annotation id="521">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:44Z</infon>
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<location offset="22447" length="8"/>
<text>subunits</text>
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<annotation id="522">
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<infon key="updated_at">2023-09-19T16:16:00Z</infon>
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<location offset="22500" length="13"/>
<text>doughnut-like</text>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<location offset="22514" length="7"/>
<text>decamer</text>
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<annotation id="524">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22564" length="7"/>
<text>Monomer</text>
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<annotation id="525">
<infon key="score">0.99908733</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22575" length="8"/>
<text>EncFtnsH</text>
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<annotation id="526">
<infon key="score">0.99880683</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:36:56Z</infon>
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<location offset="22630" length="16"/>
<text>Dimer interfaces</text>
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<annotation id="527">
<infon key="score">0.9986603</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:18Z</infon>
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<location offset="22661" length="9"/>
<text>decameric</text>
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<annotation id="528">
<infon key="score">0.99947757</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:00:10Z</infon>
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<location offset="22671" length="4"/>
<text>ring</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22679" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1886">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:44Z</infon>
<location offset="22689" length="8"/>
<text>Subunits</text>
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<infon key="score">0.99908435</infon>
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<infon key="updated_at">2023-09-19T13:37:00Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>Fe3+</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:37:03Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>Ca2+</text>
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<passage>
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<infon key="id">fig4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>22875</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.012</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>22922</offset>
<text>We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics). The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain. The protein chains were arranged as three identical annular decamers, each with D5 symmetry. The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A). The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B). The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.</text>
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<infon key="updated_at">2023-09-19T14:29:15Z</infon>
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<text>crystal structure</text>
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<location offset="22961" length="8"/>
<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T18:20:35Z</infon>
<infon key="identifier">MESH:</infon>
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<text>molecular replacement</text>
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<infon key="updated_at">2023-09-19T18:06:07Z</infon>
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<text>X-ray data collection and refinement statistics</text>
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<infon key="updated_at">2023-09-19T18:16:40Z</infon>
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<text>monomers</text>
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<infon key="updated_at">2023-09-19T11:30:39Z</infon>
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<text>EncFtn</text>
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<infon key="updated_at">2023-09-19T18:05:55Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>electron density</text>
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<infon key="updated_at">2023-09-19T13:38:12Z</infon>
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<text>7 – 96</text>
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<infon key="updated_at">2023-09-19T16:16:25Z</infon>
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<location offset="23272" length="7"/>
<text>annular</text>
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<infon key="updated_at">2023-09-19T11:53:53Z</infon>
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<location offset="23280" length="8"/>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23317" length="7"/>
<text>decamer</text>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23387" length="7"/>
<text>monomer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23398" length="6"/>
<text>EncFtn</text>
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<infon key="updated_at">2023-09-19T13:38:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23423" length="9"/>
<text>310-helix</text>
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<infon key="updated_at">2023-09-19T13:38:04Z</infon>
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<infon key="updated_at">2023-09-19T13:38:29Z</infon>
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<location offset="23542" length="7"/>
<text>helices</text>
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<infon key="updated_at">2023-09-19T13:38:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23583" length="5"/>
<text>helix</text>
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<infon key="updated_at">2023-09-19T18:00:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23612" length="2"/>
<text>α2</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:39:19Z</infon>
<location offset="23632" length="17"/>
<text>C-terminal region</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:38:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23724" length="4"/>
<text>ring</text>
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:37:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23750" length="32"/>
<text>encapsulin localization sequence</text>
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<infon key="score">0.99910885</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:20:01Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>full-length</text>
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<infon key="updated_at">2023-09-19T13:38:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23836" length="4"/>
<text>ring</text>
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<text>shell</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>23933</offset>
<text>The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D). The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain. This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C). The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D). This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.</text>
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<text>monomer</text>
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<infon key="updated_at">2023-09-19T13:13:56Z</infon>
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<text>EncFtnsH</text>
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<text>dimer interfaces</text>
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<location offset="24038" length="5"/>
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<infon key="updated_at">2023-09-19T18:16:48Z</infon>
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<location offset="24063" length="8"/>
<text>monomers</text>
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<infon key="updated_at">2023-09-19T18:00:19Z</infon>
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<location offset="24114" length="2"/>
<text>α1</text>
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<annotation id="562">
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<location offset="24127" length="7"/>
<text>monomer</text>
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<infon key="updated_at">2023-09-19T18:00:29Z</infon>
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<infon key="updated_at">2023-09-19T18:12:24Z</infon>
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<location offset="24232" length="9"/>
<text>interface</text>
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<annotation id="567">
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<infon key="updated_at">2023-09-19T18:22:58Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:23:01Z</infon>
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<infon key="updated_at">2023-09-19T11:32:02Z</infon>
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<location offset="24390" length="15"/>
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<infon key="updated_at">2023-09-19T18:00:33Z</infon>
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<location offset="24506" length="9"/>
<text>interface</text>
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<location offset="24601" length="12"/>
<text>salt bridges</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>24659</offset>
<text>The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A). Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference. The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel. The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).</text>
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<infon key="updated_at">2023-09-19T18:16:52Z</infon>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<location offset="24728" length="7"/>
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<infon key="updated_at">2023-09-19T11:30:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24739" length="6"/>
<text>EncFtn</text>
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<infon key="updated_at">2023-09-19T13:39:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24768" length="8"/>
<text>pentamer</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:37:11Z</infon>
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<location offset="24780" length="6"/>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<location offset="24805" length="7"/>
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<infon key="updated_at">2023-09-19T13:40:07Z</infon>
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<location offset="24870" length="4"/>
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<infon key="updated_at">2023-09-19T11:35:44Z</infon>
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<location offset="24910" length="8"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:40:13Z</infon>
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<location offset="24945" length="4"/>
<text>ring</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:39:46Z</infon>
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<location offset="25026" length="12"/>
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<infon key="updated_at">2023-09-19T18:06:13Z</infon>
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<infon key="updated_at">2023-09-19T13:39:56Z</infon>
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<text>central channel</text>
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<infon key="annotator">cleaner0</infon>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>25550</offset>
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<infon key="updated_at">2023-09-19T11:31:54Z</infon>
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<offset>25576</offset>
<text>Putative ligand-binding site in EncFtnsH.</text>
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<infon key="updated_at">2023-09-19T13:13:56Z</infon>
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<text>EncFtnsH</text>
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<text>(A) Wall-eyed stereo view of the dimer interface of EncFtn. Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).</text>
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<text>mFo-DFc</text>
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<infon key="updated_at">2023-09-19T13:40:58Z</infon>
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<infon key="id">fig5s1</infon>
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<infon key="type">fig_caption</infon>
<offset>26005</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.015</text>
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<passage>
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<infon key="id">fig5</infon>
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<infon key="type">fig_title_caption</infon>
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<text>EncFtnsH metal binding sites.</text>
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<infon key="updated_at">2023-09-19T14:08:39Z</infon>
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<infon key="type">fig_caption</infon>
<offset>26082</offset>
<text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains. Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains). The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.</text>
<annotation id="600">
<infon key="score">0.9990543</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:08:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26115" length="36"/>
<text>metal-binding dimerization interface</text>
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<annotation id="601">
<infon key="score">0.99433535</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="26155" length="8"/>
<text>EncFtnsH</text>
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<annotation id="602">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26248" length="8"/>
<text>subunits</text>
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<annotation id="603">
<infon key="score">0.9990736</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26258" length="4"/>
<text>iron</text>
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<annotation id="604">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26300" length="7"/>
<text>calcium</text>
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<annotation id="605">
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:24:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26333" length="13"/>
<text>glycolic acid</text>
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<annotation id="2024">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:23Z</infon>
<location offset="26410" length="14"/>
<text>di-iron center</text>
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<annotation id="606">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26430" length="29"/>
<text>2mFo-DFc electron density map</text>
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<annotation id="607">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26511" length="37"/>
<text>NCS-averaged anomalous difference map</text>
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<annotation id="608">
<infon key="score">0.9988978</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26603" length="4"/>
<text>Iron</text>
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<annotation id="2027">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:13Z</infon>
<location offset="26608" length="12"/>
<text>coordination</text>
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<annotation id="609">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26632" length="3"/>
<text>FOC</text>
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<annotation id="610">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26655" length="5"/>
<text>Glu32</text>
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<annotation id="611">
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<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26662" length="5"/>
<text>Glu62</text>
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<annotation id="612">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26669" length="5"/>
<text>His65</text>
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<annotation id="613">
<infon key="score">0.99956614</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26679" length="5"/>
<text>Tyr39</text>
</annotation>
<annotation id="2028">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:13Z</infon>
<location offset="26744" length="12"/>
<text>Coordination</text>
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<annotation id="614">
<infon key="score">0.99900657</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26781" length="4"/>
<text>iron</text>
</annotation>
<annotation id="615">
<infon key="score">0.8470298</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26853" length="12"/>
<text>Coordination</text>
</annotation>
<annotation id="616">
<infon key="score">0.9982346</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26869" length="7"/>
<text>calcium</text>
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<annotation id="617">
<infon key="score">0.99903464</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26888" length="15"/>
<text>dimer interface</text>
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<annotation id="2026">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:10:55Z</infon>
<location offset="26912" length="13"/>
<text>glutamic acid</text>
</annotation>
<annotation id="618">
<infon key="score">0.9995981</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26936" length="3"/>
<text>E31</text>
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<annotation id="619">
<infon key="score">0.9995939</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26944" length="3"/>
<text>E34</text>
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<annotation id="620">
<infon key="score">0.9987098</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26970" length="7"/>
<text>calcium</text>
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<annotation id="621">
<infon key="score">0.99776304</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:10:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27012" length="5"/>
<text>water</text>
</annotation>
<annotation id="2029">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:13Z</infon>
<location offset="27044" length="12"/>
<text>coordination</text>
</annotation>
<annotation id="622">
<infon key="score">0.99873096</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27064" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="623">
<infon key="score">0.99896955</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27102" length="23"/>
<text>Metal coordination site</text>
</annotation>
<annotation id="624">
<infon key="score">0.9538523</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27150" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="625">
<infon key="score">0.9985858</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27168" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="2056">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:19:10Z</infon>
<location offset="27185" length="14"/>
<text>coordinated by</text>
</annotation>
<annotation id="626">
<infon key="score">0.99950826</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27209" length="5"/>
<text>His57</text>
</annotation>
<annotation id="627">
<infon key="score">0.9995154</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27216" length="5"/>
<text>Glu61</text>
</annotation>
<annotation id="628">
<infon key="score">0.9995223</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27226" length="5"/>
<text>Glu64</text>
</annotation>
<annotation id="629">
<infon key="score">0.3694277</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27259" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="630">
<infon key="score">0.99882644</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:16:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27263" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="1921">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<location offset="27353" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="631">
<infon key="score">0.99894947</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27357" length="4"/>
<text>iron</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig5.jpg</infon>
<infon key="id">fig5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27371</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.014</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>27418</offset>
<text>The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1). Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data. Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains. Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A). An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization. This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B). This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin. It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27422" length="21"/>
<text>electron density maps</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27459" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2040">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:13:06Z</infon>
<location offset="27518" length="11"/>
<text>mFo-DFc map</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:13:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27551" length="14"/>
<text>4-helix bundle</text>
</annotation>
<annotation id="635">
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<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27566" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="636">
<infon key="score">0.98651385</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27620" length="6"/>
<text>ICP-MS</text>
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<annotation id="2051">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:27Z</infon>
<location offset="27647" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="637">
<infon key="score">0.99903417</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27659" length="4"/>
<text>iron</text>
</annotation>
<annotation id="2041">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:13:46Z</infon>
<location offset="27692" length="16"/>
<text>diffraction data</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27742" length="4"/>
<text>iron</text>
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<annotation id="639">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:13:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27790" length="32"/>
<text>anomalous difference Fourier map</text>
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<infon key="score">0.99651504</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:06:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27859" length="3"/>
<text>map</text>
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<annotation id="1989">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:26:00Z</infon>
<location offset="27883" length="5"/>
<text>peaks</text>
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<annotation id="641">
<infon key="score">0.9994722</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27906" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="642">
<infon key="score">0.9994836</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27913" length="5"/>
<text>Glu62</text>
</annotation>
<annotation id="643">
<infon key="score">0.9995466</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27923" length="5"/>
<text>His65</text>
</annotation>
<annotation id="644">
<infon key="score">0.99946386</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28011" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="645">
<infon key="score">0.9994611</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28021" length="5"/>
<text>Glu34</text>
</annotation>
<annotation id="2199">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:06:39Z</infon>
<location offset="28087" length="10"/>
<text>refinement</text>
</annotation>
<annotation id="646">
<infon key="score">0.9979587</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:13:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28105" length="31"/>
<text>anomalous scattering parameters</text>
</annotation>
<annotation id="647">
<infon key="score">0.99910706</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28173" length="4"/>
<text>iron</text>
</annotation>
<annotation id="648">
<infon key="score">0.9986248</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28189" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="649">
<infon key="score">0.9818403</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:06:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28262" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="650">
<infon key="score">0.9983818</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28288" length="20"/>
<text>di-iron binding site</text>
</annotation>
<annotation id="2046">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:16:46Z</infon>
<location offset="28316" length="11"/>
<text>mFo-DFc map</text>
</annotation>
<annotation id="651">
<infon key="score">0.9986642</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:24:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28343" length="13"/>
<text>glycolic acid</text>
</annotation>
<annotation id="652">
<infon key="score">0.9276309</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:16:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28396" length="8"/>
<text>PEG 3350</text>
</annotation>
<annotation id="653">
<infon key="score">0.9970285</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28436" length="14"/>
<text>di-iron center</text>
</annotation>
<annotation id="2042">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:15:27Z</infon>
<location offset="28458" length="14"/>
<text>Fe-Fe distance</text>
</annotation>
<annotation id="2043">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:15:39Z</infon>
<location offset="28483" length="18"/>
<text>Fe-Glu-O distances</text>
</annotation>
<annotation id="2044">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:15:50Z</infon>
<location offset="28529" length="18"/>
<text>Fe-His-N distances</text>
</annotation>
<annotation id="2030">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:14Z</infon>
<location offset="28575" length="12"/>
<text>coordination</text>
</annotation>
<annotation id="654">
<infon key="score">0.99805176</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:16:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28620" length="29"/>
<text>di-nuclear ferroxidase center</text>
</annotation>
<annotation id="655">
<infon key="score">0.99822277</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28651" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="656">
<infon key="score">0.98256</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28665" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="657">
<infon key="score">0.99911994</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28745" length="4"/>
<text>iron</text>
</annotation>
<annotation id="658">
<infon key="score">0.8547493</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28757" length="22"/>
<text>crystallization trials</text>
</annotation>
<annotation id="659">
<infon key="score">0.99851245</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28785" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="660">
<infon key="score">0.9989792</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28813" length="4"/>
<text>iron</text>
</annotation>
<annotation id="661">
<infon key="score">0.99712235</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:06:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28831" length="9"/>
<text>structure</text>
</annotation>
<annotation id="2045">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:16:20Z</infon>
<location offset="28882" length="8"/>
<text>affinity</text>
</annotation>
<annotation id="662">
<infon key="score">0.99907124</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28895" length="4"/>
<text>iron</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>28907</offset>
<text>The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site). A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C). While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site. The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction. A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D). These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.</text>
<annotation id="663">
<infon key="score">0.99870896</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28911" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="2057">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:19:10Z</infon>
<location offset="28923" length="14"/>
<text>coordinated by</text>
</annotation>
<annotation id="664">
<infon key="score">0.99941504</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28938" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="665">
<infon key="score">0.9994036</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28948" length="5"/>
<text>Glu34</text>
</annotation>
<annotation id="666">
<infon key="score">0.99332994</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28961" length="15"/>
<text>heptacoordinate</text>
</annotation>
<annotation id="2031">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:14Z</infon>
<location offset="28992" length="12"/>
<text>coordination</text>
</annotation>
<annotation id="667">
<infon key="score">0.99943227</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29073" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="668">
<infon key="score">0.9994186</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29083" length="5"/>
<text>Glu34</text>
</annotation>
<annotation id="669">
<infon key="score">0.9897661</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29090" length="11"/>
<text>E31/34-site</text>
</annotation>
<annotation id="670">
<infon key="score">0.6958668</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:23:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29151" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="671">
<infon key="score">0.99474263</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29210" length="15"/>
<text>heptacoordinate</text>
</annotation>
<annotation id="672">
<infon key="score">0.9982759</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:06:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29248" length="18"/>
<text>crystal structures</text>
</annotation>
<annotation id="673">
<infon key="score">0.99867094</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29272" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="674">
<infon key="score">0.9985314</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29304" length="6"/>
<text>ICP-MS</text>
</annotation>
<annotation id="675">
<infon key="score">0.9986589</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29359" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="2052">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:27Z</infon>
<location offset="29396" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="676">
<infon key="score">0.9984487</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29415" length="15"/>
<text>calcium acetate</text>
</annotation>
<annotation id="2032">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:14Z</infon>
<location offset="29479" length="12"/>
<text>coordination</text>
</annotation>
<annotation id="677">
<infon key="score">0.99859625</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29495" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="678">
<infon key="score">0.998891</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29583" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="679">
<infon key="score">0.9991187</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29595" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="680">
<infon key="score">0.99840766</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:08:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29624" length="19"/>
<text>metal binding sites</text>
</annotation>
<annotation id="681">
<infon key="score">0.9989786</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29689" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1866">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="29723" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="682">
<infon key="score">0.9987783</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29835" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="2055">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:19:09Z</infon>
<location offset="29894" length="14"/>
<text>coordinated by</text>
</annotation>
<annotation id="683">
<infon key="score">0.9994723</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29909" length="5"/>
<text>His57</text>
</annotation>
<annotation id="684">
<infon key="score">0.99945587</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29916" length="5"/>
<text>Glu61</text>
</annotation>
<annotation id="685">
<infon key="score">0.9994591</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29926" length="5"/>
<text>Glu64</text>
</annotation>
<annotation id="686">
<infon key="score">0.81935817</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="29956" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="687">
<infon key="score">0.9988262</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29960" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="2048">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:14Z</infon>
<location offset="29987" length="8"/>
<text>Fe-Glu-O</text>
</annotation>
<annotation id="2047">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:59Z</infon>
<location offset="30040" length="18"/>
<text>Fe-His-N distances</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig6-figsupp1.jpg</infon>
<infon key="id">fig6s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>30081</offset>
<text>Comparison of quaternary structure of EncFtnsH and ferritin.</text>
<annotation id="688">
<infon key="score">0.95827794</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30119" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="689">
<infon key="score">0.85408616</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30132" length="8"/>
<text>ferritin</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig6-figsupp1.jpg</infon>
<infon key="id">fig6s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>30142</offset>
<text>(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn). The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange. Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere. The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box). The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.</text>
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<infon key="updated_at">2023-09-19T14:20:38Z</infon>
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<location offset="30644" length="8"/>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.017</text>
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<passage>
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<infon key="updated_at">2023-09-19T11:28:04Z</infon>
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<infon key="updated_at">2023-09-19T11:54:09Z</infon>
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<text>(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange). Iron ions are shown as orange spheres and a single calcium ion as a grey sphere. Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue). The site in EncFtn with bound calcium is not present in other family members. (B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle. EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin. In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.</text>
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<infon key="updated_at">2023-09-19T13:13:57Z</infon>
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<location offset="30982" length="8"/>
<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<infon key="updated_at">2023-09-19T14:21:16Z</infon>
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<location offset="31022" length="28"/>
<text>Pseudo-nitzschia multiseries</text>
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<infon key="updated_at">2023-09-19T14:22:03Z</infon>
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<location offset="31051" length="8"/>
<text>ferritin</text>
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<infon key="updated_at">2023-09-19T14:20:38Z</infon>
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<location offset="31061" length="5"/>
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<infon key="updated_at">2023-09-19T11:27:31Z</infon>
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<location offset="31092" length="4"/>
<text>Iron</text>
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<infon key="updated_at">2023-09-19T14:09:59Z</infon>
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<location offset="31143" length="7"/>
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<infon key="updated_at">2023-09-19T11:54:09Z</infon>
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<location offset="31193" length="3"/>
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<infon key="updated_at">2023-09-19T14:22:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31201" length="9"/>
<text>conserved</text>
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<infon key="updated_at">2023-09-19T11:30:40Z</infon>
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<location offset="31219" length="6"/>
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<infon key="updated_at">2023-09-19T11:28:04Z</infon>
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<location offset="31230" length="8"/>
<text>ferritin</text>
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<infon key="updated_at">2023-09-19T14:22:25Z</infon>
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<location offset="31307" length="3"/>
<text>H65</text>
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<infon key="updated_at">2023-09-19T18:17:18Z</infon>
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<infon key="updated_at">2023-09-19T18:17:22Z</infon>
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<infon key="updated_at">2023-09-19T11:30:40Z</infon>
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<infon key="updated_at">2023-09-19T14:22:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31379" length="5"/>
<text>bound</text>
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<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="31385" length="7"/>
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<infon key="updated_at">2023-09-19T18:20:48Z</infon>
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<location offset="31460" length="7"/>
<text>aligned</text>
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<infon key="updated_at">2023-09-19T18:17:26Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31476" length="8"/>
<text>EncFtnsH</text>
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<annotation id="739">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31489" length="9"/>
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<infon key="updated_at">2023-09-19T11:28:04Z</infon>
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<location offset="31499" length="8"/>
<text>ferritin</text>
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<infon key="updated_at">2023-09-19T14:22:12Z</infon>
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<infon key="updated_at">2023-09-19T14:22:07Z</infon>
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<text>four-helix bundle</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
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<location offset="31554" length="8"/>
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<infon key="updated_at">2023-09-19T18:17:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31563" length="8"/>
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<infon key="updated_at">2023-09-19T18:20:51Z</infon>
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<location offset="31604" length="7"/>
<text>aligned</text>
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<infon key="score">0.9986172</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:20:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31612" length="5"/>
<text>PmFtn</text>
</annotation>
<annotation id="747">
<infon key="score">0.9989372</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31618" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="748">
<infon key="score">0.996554</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31692" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="749">
<infon key="score">0.998906</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31699" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="750">
<infon key="score">0.93538827</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31709" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="751">
<infon key="score">0.99890697</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31726" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="752">
<infon key="score">0.99850637</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31735" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="753">
<infon key="score">0.6096333</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31757" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="754">
<infon key="score">0.99891686</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31790" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="755">
<infon key="score">0.99889</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31835" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="756">
<infon key="score">0.9972924</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:23:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31884" length="11"/>
<text>long linker</text>
</annotation>
<annotation id="2060">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:23:37Z</infon>
<location offset="31904" length="2"/>
<text>α2</text>
</annotation>
<annotation id="757">
<infon key="score">0.9833993</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:23:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31911" length="2"/>
<text>α3</text>
</annotation>
<annotation id="758">
<infon key="score">0.85288185</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31917" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="759">
<infon key="score">0.99731797</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:20:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31926" length="5"/>
<text>PmFtn</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig6.jpg</infon>
<infon key="id">fig6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>31941</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.016</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>31988</offset>
<text>Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins (Figure 6A). The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A). Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B). The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).</text>
<annotation id="760">
<infon key="score">0.99876136</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31988" length="20"/>
<text>Structural alignment</text>
</annotation>
<annotation id="761">
<infon key="score">0.99906176</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32016" length="20"/>
<text>di-iron binding site</text>
</annotation>
<annotation id="762">
<infon key="score">0.9993087</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32040" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="763">
<infon key="score">0.9382234</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32056" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="764">
<infon key="score">0.95885146</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:21:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32063" length="28"/>
<text>Pseudo-nitzschia multiseries</text>
</annotation>
<annotation id="765">
<infon key="score">0.827648</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32092" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="766">
<infon key="score">0.99918133</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:20:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32102" length="5"/>
<text>PmFtn</text>
</annotation>
<annotation id="767">
<infon key="score">0.9990857</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:08:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32165" length="19"/>
<text>metal binding sites</text>
</annotation>
<annotation id="768">
<infon key="score">0.99916995</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32188" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1894">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="32205" length="9"/>
<text>classical</text>
</annotation>
<annotation id="769">
<infon key="score">0.99907744</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32215" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="770">
<infon key="score">0.99905753</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32243" length="12"/>
<text>di-iron site</text>
</annotation>
<annotation id="771">
<infon key="score">0.99903935</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32259" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="772">
<infon key="score">0.99893224</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32323" length="15"/>
<text>dimer interface</text>
</annotation>
<annotation id="773">
<infon key="score">0.9869153</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32350" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="774">
<infon key="score">0.99295735</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32357" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="775">
<infon key="score">0.9995859</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32458" length="5"/>
<text>His65</text>
</annotation>
<annotation id="776">
<infon key="score">0.99866617</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32478" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="777">
<infon key="score">0.99877733</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32485" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="778">
<infon key="score">0.9990285</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:23:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32500" length="13"/>
<text>FOC interface</text>
</annotation>
<annotation id="779">
<infon key="score">0.9994992</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32515" length="5"/>
<text>His65</text>
</annotation>
<annotation id="780">
<infon key="score">0.9987517</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:20:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32536" length="26"/>
<text>Structural superimposition</text>
</annotation>
<annotation id="781">
<infon key="score">0.985227</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:12:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32570" length="4"/>
<text>FOCs</text>
</annotation>
<annotation id="782">
<infon key="score">0.9896208</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32578" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="783">
<infon key="score">0.99907386</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32591" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="784">
<infon key="score">0.9990616</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:00:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32609" length="17"/>
<text>four-helix bundle</text>
</annotation>
<annotation id="785">
<infon key="score">0.9984042</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32634" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="786">
<infon key="score">0.99907494</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:41Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32678" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="787">
<infon key="score">0.9988463</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:17:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32685" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="788">
<infon key="score">0.9986461</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32784" length="14"/>
<text>di-iron center</text>
</annotation>
<annotation id="789">
<infon key="score">0.99873406</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:00:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32816" length="6"/>
<text>linker</text>
</annotation>
<annotation id="790">
<infon key="score">0.99882615</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32834" length="15"/>
<text>helices 2 and 3</text>
</annotation>
<annotation id="791">
<infon key="score">0.99707556</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32853" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="792">
<infon key="score">0.99891996</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32908" length="5"/>
<text>helix</text>
</annotation>
<annotation id="793">
<infon key="score">0.9969494</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:41Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32921" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="794">
<infon key="score">0.9988519</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32928" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="795">
<infon key="score">0.9993788</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32955" length="9"/>
<text>310 helix</text>
</annotation>
<annotation id="796">
<infon key="score">0.9988135</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32979" length="7"/>
<text>monomer</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>33004</offset>
<text>Mass spectrometry of the EncFtn assembly</text>
<annotation id="797">
<infon key="score">0.9986815</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:34:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33004" length="17"/>
<text>Mass spectrometry</text>
</annotation>
<annotation id="798">
<infon key="score">0.9705025</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:41Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33029" length="6"/>
<text>EncFtn</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig7-figsupp1.jpg</infon>
<infon key="id">fig7s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>33045</offset>
<text>Native IM-MS analysis of the apo-EncFtnsH monomer.</text>
<annotation id="799">
<infon key="score">0.9983001</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33045" length="12"/>
<text>Native IM-MS</text>
</annotation>
<annotation id="800">
<infon key="score">0.9993343</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33074" length="3"/>
<text>apo</text>
</annotation>
<annotation id="801">
<infon key="score">0.99771166</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33078" length="8"/>
<text>EncFtnsH</text>
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<text>(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions. The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1. The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2). The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2. Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure. It is clear from this data that apo-EncFtnsH exists in several gas phase conformations. The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large. In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez. As described by Beveridge et al., all these factors are indicative of a disordered protein.</text>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<infon key="updated_at">2023-09-19T11:32:18Z</infon>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.019</text>
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<text>The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles). The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex. This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by. In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B). This distribution is consistent with an ejected EncFtnsH dimer (orange circles). Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions. This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da]. The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding. We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions. A schematic summary of these results is displayed in (D). We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface. Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.</text>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.020</text>
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<text>Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.</text>
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<text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations. A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da). Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa). (B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer. Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa). Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th. The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer. Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted. The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).</text>
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<text>holo</text>
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<infon key="updated_at">2023-09-19T14:25:50Z</infon>
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<text>charge states</text>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.018</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>39177</offset>
<text>In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS). As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron. Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da). Under these conditions, no significant higher order assembly was observed and the protein did not have any coordinated metal ions. Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3). After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa). MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry. Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site. These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage. Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B). This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer. By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure. Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies. Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.</text>
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<infon key="updated_at">2023-09-19T14:35:40Z</infon>
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<infon key="updated_at">2023-09-19T14:35:44Z</infon>
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<infon key="updated_at">2023-09-19T11:53:46Z</infon>
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<location offset="40283" length="7"/>
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<location offset="40341" length="11"/>
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<infon key="updated_at">2023-09-19T16:25:03Z</infon>
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<location offset="40356" length="5"/>
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<infon key="updated_at">2023-09-19T11:54:09Z</infon>
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<location offset="40369" length="3"/>
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<infon key="updated_at">2023-09-19T14:09:59Z</infon>
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<location offset="40435" length="7"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
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<infon key="updated_at">2023-09-19T14:36:04Z</infon>
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<location offset="40469" length="5"/>
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<infon key="updated_at">2023-09-19T14:35:57Z</infon>
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<location offset="40496" length="2"/>
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<infon key="updated_at">2023-09-19T14:35:47Z</infon>
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<infon key="updated_at">2023-09-19T11:27:31Z</infon>
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<infon key="updated_at">2023-09-19T11:27:31Z</infon>
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<infon key="updated_at">2023-09-19T11:32:19Z</infon>
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<infon key="updated_at">2023-09-19T11:27:31Z</infon>
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<infon key="updated_at">2023-09-19T11:28:05Z</infon>
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<location offset="40828" length="8"/>
<text>ferritin</text>
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<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
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<location offset="40898" length="8"/>
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<infon key="updated_at">2023-09-19T18:21:17Z</infon>
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<location offset="40908" length="21"/>
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<infon key="updated_at">2023-09-19T11:32:19Z</infon>
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<infon key="updated_at">2023-09-19T14:36:12Z</infon>
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<infon key="updated_at">2023-09-19T14:27:51Z</infon>
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<text>CCS</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:27:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="41180" length="3"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-19T14:35:00Z</infon>
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<infon key="updated_at">2023-09-19T18:18:48Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="identifier">PR:</infon>
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<infon key="updated_at">2023-09-19T16:16:56Z</infon>
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<infon key="updated_at">2023-09-19T16:17:00Z</infon>
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<location offset="41380" length="10"/>
<text>metal-free</text>
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<infon key="updated_at">2023-09-19T18:11:54Z</infon>
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<infon key="updated_at">2023-09-19T13:13:40Z</infon>
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<infon key="identifier">DUMMY:</infon>
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<text>IM-MS</text>
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<location offset="41765" length="8"/>
<text>EncFtnsH</text>
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<text>4-helix bundle</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:02Z</infon>
<location offset="42011" length="15"/>
<text>dimer interface</text>
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<annotation id="1896">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="42059" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1840">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="42069" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="2101">
<infon key="type">oligomeric_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:37:10Z</infon>
<location offset="42112" length="6"/>
<text>dimers</text>
</annotation>
<annotation id="1865">
<infon key="type">oligomeric_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<location offset="42187" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="2091">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<location offset="42230" length="17"/>
<text>crystal structure</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>42254</offset>
<text>We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry. Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation. For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2). In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe. Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface. In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.</text>
<annotation id="984">
<infon key="score">0.9986589</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42309" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="985">
<infon key="score">0.99868244</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42319" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="986">
<infon key="score">0.9986521</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42334" length="30"/>
<text>collision-induced dissociation</text>
</annotation>
<annotation id="987">
<infon key="score">0.99799937</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:31:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42366" length="3"/>
<text>CID</text>
</annotation>
<annotation id="988">
<infon key="score">0.97773576</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:37:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42371" length="24"/>
<text>tandem mass spectrometry</text>
</annotation>
<annotation id="989">
<infon key="score">0.9944747</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:31:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42415" length="3"/>
<text>CID</text>
</annotation>
<annotation id="990">
<infon key="score">0.9986993</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42662" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="991">
<infon key="score">0.99808913</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:31:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="42728" length="3"/>
<text>CID</text>
</annotation>
<annotation id="992">
<infon key="score">0.9966664</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42739" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="993">
<infon key="score">0.99880517</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42748" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="994">
<infon key="score">0.998884</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42788" length="7"/>
<text>dimeric</text>
</annotation>
<annotation id="995">
<infon key="score">0.99784243</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42796" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="996">
<infon key="score">0.99911135</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="42838" length="4"/>
<text>iron</text>
</annotation>
<annotation id="997">
<infon key="score">0.9988296</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42896" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="998">
<infon key="score">0.99771583</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="42975" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="999">
<infon key="score">0.9988618</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="42984" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1000">
<infon key="score">0.99905413</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:38:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43013" length="17"/>
<text>non-FOC interface</text>
</annotation>
<annotation id="1001">
<infon key="score">0.99905515</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:38:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43070" length="13"/>
<text>FOC interface</text>
</annotation>
<annotation id="1923">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<location offset="43092" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="2102">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:38:54Z</infon>
<location offset="43096" length="2"/>
<text>Fe</text>
</annotation>
<annotation id="1002">
<infon key="score">0.9985984</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43195" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1003">
<infon key="score">0.99854517</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43218" length="10"/>
<text>pentameric</text>
</annotation>
<annotation id="1004">
<infon key="score">0.99877137</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:37:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43241" length="6"/>
<text>dimers</text>
</annotation>
<annotation id="1005">
<infon key="score">0.99917513</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="43257" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1006">
<infon key="score">0.9990354</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="43282" length="19"/>
<text>FOC dimer interface</text>
</annotation>
<annotation id="1007">
<infon key="score">0.84026235</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43414" length="7"/>
<text>crystal</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>43454</offset>
<text>Ferroxidase activity</text>
<annotation id="1008">
<infon key="score">0.9968352</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="43454" length="11"/>
<text>Ferroxidase</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig8-figsupp1.jpg</infon>
<infon key="id">fig8s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>43475</offset>
<text>TEM visualization of iron-loaded bacterial nanocompartments and ferritin.</text>
<annotation id="1009">
<infon key="score">0.99874395</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="43475" length="3"/>
<text>TEM</text>
</annotation>
<annotation id="2103">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:34Z</infon>
<location offset="43496" length="11"/>
<text>iron-loaded</text>
</annotation>
<annotation id="1010">
<infon key="score">0.9987717</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43508" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="1011">
<infon key="score">0.99808806</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="43518" length="16"/>
<text>nanocompartments</text>
</annotation>
<annotation id="1012">
<infon key="score">0.65248877</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="43539" length="8"/>
<text>ferritin</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig8-figsupp1.jpg</infon>
<infon key="id">fig8s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>43549</offset>
<text>Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively. Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain. (A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.</text>
<annotation id="1013">
<infon key="score">0.9983839</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43549" length="9"/>
<text>Decameric</text>
</annotation>
<annotation id="1014">
<infon key="score">0.99588805</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43559" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1015">
<infon key="score">0.99626344</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43569" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1016">
<infon key="score">0.721113</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:07Z</infon>
<infon key="identifier">GO:</infon>
<location offset="43581" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1017">
<infon key="score">0.98189974</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43596" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1967">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:56Z</infon>
<location offset="43673" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
</annotation>
<annotation id="1018">
<infon key="score">0.998538</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="43773" length="3"/>
<text>TEM</text>
</annotation>
<annotation id="1019">
<infon key="score">0.92946935</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:40:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="43802" length="14"/>
<text>uranyl acetate</text>
</annotation>
<annotation id="1020">
<infon key="score">0.9812164</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43840" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1021">
<infon key="score">0.99050516</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="43850" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1022">
<infon key="score">0.8156171</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:07Z</infon>
<infon key="identifier">GO:</infon>
<location offset="43862" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1023">
<infon key="score">0.9878298</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43874" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1024">
<infon key="score">0.9927024</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="43886" length="11"/>
<text>loaded with</text>
</annotation>
<annotation id="2107">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:40:41Z</infon>
<location offset="43898" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1025">
<infon key="score">0.719513</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:17:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="44022" length="7"/>
<text>Stained</text>
</annotation>
<annotation id="1026">
<infon key="score">0.99073374</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44030" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1027">
<infon key="score">0.9944073</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="44040" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1028">
<infon key="score">0.74889445</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="44052" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1029">
<infon key="score">0.99222344</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44064" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1030">
<infon key="score">0.99380374</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="44076" length="11"/>
<text>loaded with</text>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.022</text>
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<text>Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.</text>
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<text>(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer. The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer. Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin. The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.</text>
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<text>Encapsulin</text>
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<infon key="updated_at">2023-09-19T12:00:08Z</infon>
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<text>EncFtn-Enc</text>
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<text>incubated</text>
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<text>iron</text>
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<infon key="updated_at">2023-09-19T14:42:11Z</infon>
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<text>UV/visible spectrophotometer</text>
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<infon key="updated_at">2023-09-19T14:42:29Z</infon>
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<text>Fe2+</text>
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<infon key="updated_at">2023-09-19T11:27:32Z</infon>
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<text>iron</text>
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<text>EncFtnsH</text>
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<location offset="45167" length="10"/>
<text>encapsulin</text>
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<infon key="updated_at">2023-09-19T12:00:08Z</infon>
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<location offset="45179" length="10"/>
<text>EncFtn-Enc</text>
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<infon key="updated_at">2023-09-19T16:21:26Z</infon>
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<text>apoferritin</text>
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<infon key="updated_at">2023-09-19T11:27:32Z</infon>
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<offset>45367</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.021</text>
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<passage>
<infon key="section_type">RESULTS</infon>
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<offset>45414</offset>
<text>In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein. In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control. Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine. The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC). In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A). These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme. When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment. Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction. We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B). In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).</text>
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<infon key="updated_at">2023-09-19T11:30:41Z</infon>
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<infon key="updated_at">2023-09-19T11:55:10Z</infon>
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<text>native mass spectrometry</text>
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<infon key="updated_at">2023-09-19T14:43:23Z</infon>
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<infon key="updated_at">2023-09-19T14:43:31Z</infon>
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<text>equine</text>
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<infon key="updated_at">2023-09-19T16:21:26Z</infon>
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<text>apoferritin</text>
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<text>classical</text>
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<infon key="updated_at">2023-09-19T11:28:05Z</infon>
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<text>ferritin</text>
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<infon key="updated_at">2023-09-19T14:44:50Z</infon>
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<text>Dps family</text>
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<infon key="updated_at">2023-09-19T14:44:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="45783" length="22"/>
<text>ferritin-like proteins</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="45807" length="6"/>
<text>EncFtn</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="45876" length="22"/>
<text>ortho-phenylenediamine</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
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<text>ferroxidase</text>
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<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
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<text>EncFtnsH</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:44:56Z</infon>
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<text>progress curve</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="45985" length="4"/>
<text>Fe2+</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46003" length="4"/>
<text>Fe3+</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:58Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46050" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1080">
<infon key="score">0.9985999</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46083" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1081">
<infon key="score">0.7469009</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="46088" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1082">
<infon key="score">0.9989803</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:43:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46176" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1884">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:28Z</infon>
<location offset="46194" length="6"/>
<text>oxygen</text>
</annotation>
<annotation id="1083">
<infon key="score">0.99907494</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:44:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46229" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1084">
<infon key="score">0.99886835</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="46280" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1085">
<infon key="score">0.9981989</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="46300" length="6"/>
<text>active</text>
</annotation>
<annotation id="1086">
<infon key="score">0.9988611</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="46307" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="2162">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:25Z</infon>
<location offset="46344" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1087">
<infon key="score">0.99920136</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="46357" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1088">
<infon key="score">0.99899256</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:44:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46375" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1089">
<infon key="score">0.998909</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46508" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1090">
<infon key="score">0.99877864</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="46650" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1869">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="46705" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="1091">
<infon key="score">0.99899435</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:44:09Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46743" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1092">
<infon key="score">0.9947719</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:44:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="46801" length="4"/>
<text>full</text>
</annotation>
<annotation id="1093">
<infon key="score">0.47335646</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="46806" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1094">
<infon key="score">0.79352003</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="46813" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1095">
<infon key="score">0.5890131</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:02Z</infon>
<infon key="identifier">GO:</infon>
<location offset="46824" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1096">
<infon key="score">0.9988458</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="46850" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1870">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="46888" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="1097">
<infon key="score">0.98846835</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="46923" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>46994</offset>
<text>We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form. This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.</text>
<annotation id="2109">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:47:33Z</infon>
<location offset="47041" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2110">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:47:45Z</infon>
<location offset="47050" length="17"/>
<text>ferroxidase assay</text>
</annotation>
<annotation id="1098">
<infon key="score">0.997488</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47099" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1099">
<infon key="score">0.6162079</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47144" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1100">
<infon key="score">0.9987997</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47175" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1101">
<infon key="score">0.9978562</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:35:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="47228" length="9"/>
<text>native MS</text>
</annotation>
<annotation id="1102">
<infon key="score">0.9989479</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47273" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1103">
<infon key="score">0.99900645</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47295" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1104">
<infon key="score">0.9984871</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="47309" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="1105">
<infon key="score">0.9560211</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47319" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1106">
<infon key="score">0.9984837</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:08:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="47335" length="9"/>
<text>structure</text>
</annotation>
<annotation id="1107">
<infon key="score">0.99901986</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="47382" length="19"/>
<text>iron-storage cavity</text>
</annotation>
<annotation id="1898">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="47420" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1108">
<infon key="score">0.99909246</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="47430" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1109">
<infon key="score">0.9692733</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="47444" length="19"/>
<text>Dps family proteins</text>
</annotation>
<annotation id="1110">
<infon key="score">0.9987999</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:45:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47501" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1111">
<infon key="score">0.98059124</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:06Z</infon>
<infon key="identifier">GO:</infon>
<location offset="47519" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1112">
<infon key="score">0.95718</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:08:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="47535" length="10"/>
<text>structures</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>47549</offset>
<text>To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay. The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A). A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B). In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment. Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D). Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).</text>
<annotation id="1113">
<infon key="score">0.9982615</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47618" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1114">
<infon key="score">0.99852943</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47629" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1115">
<infon key="score">0.9990024</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47659" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1116">
<infon key="score">0.99870753</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="47696" length="3"/>
<text>TEM</text>
</annotation>
<annotation id="1117">
<infon key="score">0.99861383</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:46:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="47734" length="17"/>
<text>ferroxidase assay</text>
</annotation>
<annotation id="1118">
<infon key="score">0.9622654</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47757" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1119">
<infon key="score">0.99917233</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:46:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="47952" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1120">
<infon key="score">0.9985311</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="47964" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1121">
<infon key="score">0.9991504</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:46:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48041" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1122">
<infon key="score">0.99607974</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48053" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1123">
<infon key="score">0.9917857</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:10Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48064" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1124">
<infon key="score">0.9989164</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48271" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1125">
<infon key="score">0.9860162</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:14Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48287" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1126">
<infon key="score">0.99915993</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:46:29Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48316" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1127">
<infon key="score">0.9991714</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="48324" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1128">
<infon key="score">0.99494594</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="48451" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1129">
<infon key="score">0.99889404</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48497" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1130">
<infon key="score">0.99875206</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:59:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="48535" length="18"/>
<text>Negative stain TEM</text>
</annotation>
<annotation id="1131">
<infon key="score">0.9991665</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48602" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1132">
<infon key="score">0.9977938</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="48612" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1133">
<infon key="score">0.9987331</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="48703" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1134">
<infon key="score">0.99697083</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48715" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1135">
<infon key="score">0.999108</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="48738" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1136">
<infon key="score">0.97162443</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48774" length="16"/>
<text>nanocompartments</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>48882</offset>
<text>Iron storage in encapsulin nanocompartments</text>
<annotation id="1137">
<infon key="score">0.99887663</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="48882" length="4"/>
<text>Iron</text>
</annotation>
<annotation id="1138">
<infon key="score">0.9917041</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="48898" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1139">
<infon key="score">0.99208003</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="48909" length="16"/>
<text>nanocompartments</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>48926</offset>
<text>The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage. To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation. The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C). In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation. The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions. This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).</text>
<annotation id="1140">
<infon key="score">0.99855113</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:47:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="48945" length="17"/>
<text>ferroxidase assay</text>
</annotation>
<annotation id="1141">
<infon key="score">0.9556643</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:46:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="48967" length="11"/>
<text>micrographs</text>
</annotation>
<annotation id="1899">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="49066" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1142">
<infon key="score">0.9991431</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="49076" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1143">
<infon key="score">0.99826056</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49108" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1144">
<infon key="score">0.9960299</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49119" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1145">
<infon key="score">0.9961708</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49149" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1146">
<infon key="score">0.9989749</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="49168" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="1147">
<infon key="score">0.59291255</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49188" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1148">
<infon key="score">0.9942485</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="49199" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1149">
<infon key="score">0.99858165</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49247" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1150">
<infon key="score">0.9991892</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:25:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49318" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1151">
<infon key="score">0.9993333</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="49363" length="3"/>
<text>apo</text>
</annotation>
<annotation id="1152">
<infon key="score">0.98954636</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="49367" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1153">
<infon key="score">0.998869</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49377" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1154">
<infon key="score">0.99516773</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49394" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1155">
<infon key="score">0.998946</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="49414" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1156">
<infon key="score">0.9985255</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="49475" length="15"/>
<text>ferrozine assay</text>
</annotation>
<annotation id="1157">
<infon key="score">0.9990097</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49517" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1158">
<infon key="score">0.99884796</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49596" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1159">
<infon key="score">0.9990859</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49669" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1160">
<infon key="score">0.9920364</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49730" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1161">
<infon key="score">0.9977925</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="49739" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1162">
<infon key="score">0.9991054</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49776" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1163">
<infon key="score">0.9988959</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49800" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1164">
<infon key="score">0.9298189</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="49840" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1165">
<infon key="score">0.9822818</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="49851" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1166">
<infon key="score">0.9990381</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="49890" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1167">
<infon key="score">0.9988372</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="49962" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="2186">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:16Z</infon>
<location offset="49973" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1168">
<infon key="score">0.99901927</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="50012" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1169">
<infon key="score">0.9975051</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50073" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1170">
<infon key="score">0.99898714</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="50139" length="4"/>
<text>iron</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>50186</offset>
<text>Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization. EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis. The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D). These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins. While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5). This implies that the shell can bind a significant amount of iron on its outer and inner surfaces. While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions. However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin. We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.</text>
<annotation id="1171">
<infon key="score">0.9991943</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="50261" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1172">
<infon key="score">0.9986137</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50273" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1173">
<infon key="score">0.9991689</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="50285" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1174">
<infon key="score">0.62141466</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:36:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50305" length="11"/>
<text>icosahedral</text>
</annotation>
<annotation id="1175">
<infon key="score">0.36811516</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:01:23Z</infon>
<infon key="identifier">GO:</infon>
<location offset="50317" length="4"/>
<text>cage</text>
</annotation>
<annotation id="1176">
<infon key="score">0.9982237</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="50345" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1177">
<infon key="score">0.9862146</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="50437" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1178">
<infon key="score">0.9909506</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="50470" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1179">
<infon key="score">0.9985505</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50477" length="8"/>
<text>decamers</text>
</annotation>
<annotation id="1180">
<infon key="score">0.9991522</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="50500" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1181">
<infon key="score">0.9950546</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="50613" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1182">
<infon key="score">0.998394</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="50621" length="11"/>
<text>E31/34-site</text>
</annotation>
<annotation id="1183">
<infon key="score">0.99747866</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50640" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="1184">
<infon key="score">0.97772205</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="50665" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1185">
<infon key="score">0.9986551</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="50674" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1186">
<infon key="score">0.9938304</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:48:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="50690" length="2"/>
<text>MS</text>
</annotation>
<annotation id="1187">
<infon key="score">0.99414957</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="50733" length="21"/>
<text>solution measurements</text>
</annotation>
<annotation id="1188">
<infon key="score">0.9964463</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:48:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="50763" length="2"/>
<text>MS</text>
</annotation>
<annotation id="1189">
<infon key="score">0.9990403</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:48:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="50814" length="19"/>
<text>metal-binding sites</text>
</annotation>
<annotation id="1190">
<infon key="score">0.9957569</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:49:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="50850" length="7"/>
<text>channel</text>
</annotation>
<annotation id="1191">
<infon key="score">0.92116725</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:13:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="50956" length="19"/>
<text>metal-binding sites</text>
</annotation>
<annotation id="1192">
<infon key="score">0.9982085</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="51013" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="1193">
<infon key="score">0.9989925</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:49:16Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51092" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1194">
<infon key="score">0.9990625</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:13:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="51125" length="11"/>
<text>active site</text>
</annotation>
<annotation id="1195">
<infon key="score">0.99875194</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:17:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="51146" length="11"/>
<text>E31/34-site</text>
</annotation>
<annotation id="1196">
<infon key="score">0.99796826</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:54:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51185" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1197">
<infon key="score">0.99880934</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="51241" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1198">
<infon key="score">0.9989153</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="51286" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1199">
<infon key="score">0.99870276</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="51297" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1871">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="51324" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="1200">
<infon key="score">0.9991266</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51367" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1201">
<infon key="score">0.9986784</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="51423" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1202">
<infon key="score">0.99907243</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51462" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1900">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="51547" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1203">
<infon key="score">0.9990132</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="51557" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1204">
<infon key="score">0.9991204</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51589" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1205">
<infon key="score">0.99912053</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="51646" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1206">
<infon key="score">0.99909496</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51674" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1207">
<infon key="score">0.99617934</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="51710" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1208">
<infon key="score">0.98968834</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:20Z</infon>
<infon key="identifier">GO:</infon>
<location offset="51721" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1209">
<infon key="score">0.99910504</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51764" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1210">
<infon key="score">0.99843305</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="51840" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1211">
<infon key="score">0.99742645</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51902" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1212">
<infon key="score">0.9990716</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="51919" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="1213">
<infon key="score">0.99191284</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="51955" length="4"/>
<text>iron</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>52055</offset>
<text>Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites. In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron. Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins. Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.</text>
<annotation id="1214">
<infon key="score">0.9989417</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52090" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1215">
<infon key="score">0.9990474</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:49:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52123" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1216">
<infon key="score">0.99900883</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:49:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52131" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1217">
<infon key="score">0.9990871</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52146" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1218">
<infon key="score">0.9989825</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52162" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1219">
<infon key="score">0.58354443</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="52187" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1220">
<infon key="score">0.97698575</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:08:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="52211" length="19"/>
<text>metal binding sites</text>
</annotation>
<annotation id="1221">
<infon key="score">0.9950051</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52249" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1222">
<infon key="score">0.9990188</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52353" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1223">
<infon key="score">0.9991837</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="52372" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1224">
<infon key="score">0.86663836</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:57:24Z</infon>
<infon key="identifier">GO:</infon>
<location offset="52383" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1225">
<infon key="score">0.9991554</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52441" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1226">
<infon key="score">0.99898523</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52464" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1227">
<infon key="score">0.75144476</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52480" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1228">
<infon key="score">0.9940952</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="52491" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1901">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="52604" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1229">
<infon key="score">0.9992168</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="52614" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1230">
<infon key="score">0.99845505</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="52654" length="16"/>
<text>nanocompartments</text>
</annotation>
<annotation id="1231">
<infon key="score">0.99830496</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="52733" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1232">
<infon key="score">0.8595094</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="52758" length="6"/>
<text>native</text>
</annotation>
<annotation id="1233">
<infon key="score">0.9983076</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="52765" length="16"/>
<text>nanocompartments</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>52843</offset>
<text>Mutagenesis of the EncFtnsHferroxidase center</text>
<annotation id="1234">
<infon key="score">0.998384</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="52843" length="11"/>
<text>Mutagenesis</text>
</annotation>
<annotation id="2111">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:50:35Z</infon>
<location offset="52862" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2112">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:50:48Z</infon>
<location offset="52870" length="18"/>
<text>ferroxidase center</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig9.jpg</infon>
<infon key="id">fig9</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>52889</offset>
<text>Purification of recombinant R. rubrum EncFtnsH FOC mutants.</text>
<annotation id="1930">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:56:17Z</infon>
<location offset="52917" length="9"/>
<text>R. rubrum</text>
</annotation>
<annotation id="1957">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<location offset="52927" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1235">
<infon key="score">0.66427743</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="52936" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="2119">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="52940" length="7"/>
<text>mutants</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig9.jpg</infon>
<infon key="id">fig9</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>52949</offset>
<text>Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography. (A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.</text>
<annotation id="2120">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="52956" length="7"/>
<text>mutants</text>
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<annotation id="1236">
<infon key="score">0.9984068</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="52964" length="4"/>
<text>E32A</text>
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<infon key="score">0.99859613</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="52970" length="4"/>
<text>E62A</text>
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<annotation id="1238">
<infon key="score">0.99884856</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="52980" length="4"/>
<text>H65A</text>
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<infon key="score">0.99898475</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="52988" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2113">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:34Z</infon>
<location offset="53011" length="17"/>
<text>E. coli BL21(DE3)</text>
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<annotation id="2014">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="53044" length="2"/>
<text>MM</text>
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<annotation id="2015">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="53051" length="2"/>
<text>MM</text>
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<annotation id="1240">
<infon key="score">0.9990044</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="53072" length="4"/>
<text>iron</text>
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<annotation id="2114">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:51Z</infon>
<location offset="53108" length="29"/>
<text>size-exclusion chromatography</text>
</annotation>
<annotation id="1241">
<infon key="score">0.9805692</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:50:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53143" length="27"/>
<text>Gel-filtration chromatogram</text>
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<annotation id="1242">
<infon key="score">0.99885917</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53178" length="4"/>
<text>E32A</text>
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<annotation id="1243">
<infon key="score">0.9984256</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53183" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1244">
<infon key="score">0.99881625</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53198" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2116">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:13Z</infon>
<location offset="53222" length="15"/>
<text>elution profile</text>
</annotation>
<annotation id="1245">
<infon key="score">0.9986394</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53284" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="1246">
<infon key="score">0.99889296</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53340" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1247">
<infon key="score">0.99896735</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53353" length="28"/>
<text>Gel-filtration chromatograhy</text>
</annotation>
<annotation id="1248">
<infon key="score">0.99888855</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53389" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1249">
<infon key="score">0.998744</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53394" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1250">
<infon key="score">0.9989052</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53409" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2118">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:22Z</infon>
<location offset="53433" length="15"/>
<text>elution profile</text>
</annotation>
<annotation id="1251">
<infon key="score">0.9985852</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53469" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="1252">
<infon key="score">0.99894845</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53489" length="29"/>
<text>Gel-filtration chromatography</text>
</annotation>
<annotation id="1253">
<infon key="score">0.99890745</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53526" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1254">
<infon key="score">0.9987801</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53531" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1255">
<infon key="score">0.9989441</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53546" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1256">
<infon key="score">0.9988803</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53610" length="7"/>
<text>monomer</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig9.jpg</infon>
<infon key="id">fig9</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>53626</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.023</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>53673</offset>
<text>To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65. These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron. The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9). For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.</text>
<annotation id="1257">
<infon key="score">0.99893826</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:54:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="53738" length="22"/>
<text>metal binding residues</text>
</annotation>
<annotation id="2115">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:54:25Z</infon>
<location offset="53768" length="11"/>
<text>di-iron FOC</text>
</annotation>
<annotation id="1258">
<infon key="score">0.9993605</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53783" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1259">
<infon key="score">0.99388635</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:54:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53804" length="17"/>
<text>alanine mutations</text>
</annotation>
<annotation id="1260">
<infon key="score">0.9995384</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53849" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="1261">
<infon key="score">0.99954516</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53856" length="5"/>
<text>Glu62</text>
</annotation>
<annotation id="1262">
<infon key="score">0.9995535</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:24:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53867" length="5"/>
<text>His65</text>
</annotation>
<annotation id="1263">
<infon key="score">0.99760395</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="53880" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1264">
<infon key="score">0.9511125</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53889" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1265">
<infon key="score">0.99831694</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:56:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53914" length="7"/>
<text>E. coli</text>
</annotation>
<annotation id="2016">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:30:27Z</infon>
<location offset="53937" length="2"/>
<text>MM</text>
</annotation>
<annotation id="1266">
<infon key="score">0.96619594</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:54:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53953" length="7"/>
<text>absence</text>
</annotation>
<annotation id="1267">
<infon key="score">0.78286123</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="53965" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="1268">
<infon key="score">0.9992204</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="53988" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1269">
<infon key="score">0.99906474</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="53998" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1270">
<infon key="score">0.99910444</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54007" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1271">
<infon key="score">0.9685832</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54012" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1272">
<infon key="score">0.9986475</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:18:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54032" length="3"/>
<text>SEC</text>
</annotation>
<annotation id="1273">
<infon key="score">0.9986431</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54068" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="1274">
<infon key="score">0.999308</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54086" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1275">
<infon key="score">0.9988231</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54123" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1276">
<infon key="score">0.9990706</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54136" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1277">
<infon key="score">0.9901754</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54141" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1278">
<infon key="score">0.9987929</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54187" length="9"/>
<text>monomeric</text>
</annotation>
<annotation id="1279">
<infon key="score">0.99932194</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54205" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2121">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="54241" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1280">
<infon key="score">0.9992074</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:54:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54323" length="4"/>
<text>Fe2+</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig10.jpg</infon>
<infon key="id">fig10</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>54339</offset>
<text>Native mass spectrometry of EncFtnsH mutants.</text>
<annotation id="1281">
<infon key="score">0.9988263</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:55:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54339" length="24"/>
<text>Native mass spectrometry</text>
</annotation>
<annotation id="1282">
<infon key="score">0.8879092</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54367" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1283">
<infon key="score">0.6093207</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54376" length="7"/>
<text>mutants</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig10.jpg</infon>
<infon key="id">fig10</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>54385</offset>
<text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles). (C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).</text>
<annotation id="1284">
<infon key="score">0.93247825</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:08:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54389" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="1285">
<infon key="score">0.5322182</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:54:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54430" length="7"/>
<text>acetate</text>
</annotation>
<annotation id="1286">
<infon key="score">0.9991552</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54488" length="9"/>
<text>Wild-type</text>
</annotation>
<annotation id="1287">
<infon key="score">0.99839765</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54498" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1288">
<infon key="score">0.998904</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54514" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1289">
<infon key="score">0.9989849</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="54525" length="4"/>
<text>iron</text>
</annotation>
<annotation id="2133">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:56:17Z</infon>
<location offset="54541" length="25"/>
<text>charge state distribution</text>
</annotation>
<annotation id="1290">
<infon key="score">0.9988834</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54585" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1291">
<infon key="score">0.99896574</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54618" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1958">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<location offset="54623" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2067">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:50Z</infon>
<location offset="54643" length="13"/>
<text>charge states</text>
</annotation>
<annotation id="1292">
<infon key="score">0.9988796</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54675" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1293">
<infon key="score">0.9989023</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54737" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1294">
<infon key="score">0.99907815</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54748" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1295">
<infon key="score">0.99743974</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54753" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1296">
<infon key="score">0.9990503</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54797" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1297">
<infon key="score">0.48325944</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54802" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2068">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:50Z</infon>
<location offset="54820" length="13"/>
<text>charge states</text>
</annotation>
<annotation id="1298">
<infon key="score">0.9988966</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54852" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1299">
<infon key="score">0.9989421</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="54881" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1300">
<infon key="score">0.5852406</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="54886" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2069">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:50Z</infon>
<location offset="54904" length="13"/>
<text>charge states</text>
</annotation>
<annotation id="1301">
<infon key="score">0.9989104</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54939" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1302">
<infon key="score">0.99890864</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="54966" length="5"/>
<text>dimer</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig10.jpg</infon>
<infon key="id">fig10</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>54994</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.024</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>55041</offset>
<text>In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10). As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron. Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+. This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution. Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC. In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.</text>
<annotation id="1303">
<infon key="score">0.99877673</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:18:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55056" length="3"/>
<text>SEC</text>
</annotation>
<annotation id="1304">
<infon key="score">0.99891645</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:55:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55069" length="24"/>
<text>native mass spectrometry</text>
</annotation>
<annotation id="1305">
<infon key="score">0.9993616</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55101" length="3"/>
<text>apo</text>
</annotation>
<annotation id="1306">
<infon key="score">0.99814653</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55105" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2122">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="55114" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1307">
<infon key="score">0.9991128</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55158" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1308">
<infon key="score">0.99933857</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55168" length="3"/>
<text>apo</text>
</annotation>
<annotation id="1309">
<infon key="score">0.99902344</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55172" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1310">
<infon key="score">0.99935716</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55226" length="3"/>
<text>apo</text>
</annotation>
<annotation id="1311">
<infon key="score">0.9991222</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55230" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2089">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:26:53Z</infon>
<location offset="55245" length="12"/>
<text>charge state</text>
</annotation>
<annotation id="1312">
<infon key="score">0.99890757</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55290" length="12"/>
<text>unstructured</text>
</annotation>
<annotation id="1313">
<infon key="score">0.9989256</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55303" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1314">
<infon key="score">0.9987863</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55316" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1315">
<infon key="score">0.9796138</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55377" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1316">
<infon key="score">0.9990503</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55392" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1317">
<infon key="score">0.99865746</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55397" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1318">
<infon key="score">0.9990827</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55408" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1319">
<infon key="score">0.99803156</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55413" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="2090">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:26:53Z</infon>
<location offset="55430" length="12"/>
<text>charge state</text>
</annotation>
<annotation id="1320">
<infon key="score">0.9987509</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55473" length="8"/>
<text>decamers</text>
</annotation>
<annotation id="1321">
<infon key="score">0.9990344</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55495" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1322">
<infon key="score">0.9990151</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:56:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55506" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1323">
<infon key="score">0.998831</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:18:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55558" length="3"/>
<text>SEC</text>
</annotation>
<annotation id="1324">
<infon key="score">0.9987618</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55624" length="8"/>
<text>decamers</text>
</annotation>
<annotation id="1325">
<infon key="score">0.9988433</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55691" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1326">
<infon key="score">0.9988737</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55714" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1327">
<infon key="score">0.9989624</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="55725" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1924">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<location offset="55737" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="2134">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:57:17Z</infon>
<location offset="55761" length="13"/>
<text>glutamic acid</text>
</annotation>
<annotation id="2123">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="55775" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1328">
<infon key="score">0.9985696</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55784" length="2"/>
<text>MS</text>
</annotation>
<annotation id="1329">
<infon key="score">0.99906534</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55803" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1330">
<infon key="score">0.99826705</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55808" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1331">
<infon key="score">0.999105</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55829" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1332">
<infon key="score">0.9993519</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:25:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55839" length="3"/>
<text>apo</text>
</annotation>
<annotation id="1333">
<infon key="score">0.9991072</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="55843" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1334">
<infon key="score">0.998919</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55872" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1335">
<infon key="score">0.99894994</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="55917" length="7"/>
<text>dimeric</text>
</annotation>
<annotation id="1336">
<infon key="score">0.9987979</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="55925" length="4"/>
<text>H65A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>55950</offset>
<text>We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC. At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state. In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron. Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC. The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge. The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.</text>
<annotation id="1337">
<infon key="score">0.99901664</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56027" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1338">
<infon key="score">0.99910337</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56036" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1339">
<infon key="score">0.9096816</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56041" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1340">
<infon key="score">0.9989551</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56061" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1341">
<infon key="score">0.91614443</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56138" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1342">
<infon key="score">0.9901097</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:57:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56146" length="10"/>
<text>neutral pH</text>
</annotation>
<annotation id="2135">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:58:05Z</infon>
<location offset="56161" length="13"/>
<text>glutamic acid</text>
</annotation>
<annotation id="1343">
<infon key="score">0.997472</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:22:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56218" length="9"/>
<text>histidine</text>
</annotation>
<annotation id="1344">
<infon key="score">0.9990551</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56288" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1345">
<infon key="score">0.99926955</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:57:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56299" length="2"/>
<text>WT</text>
</annotation>
<annotation id="1346">
<infon key="score">0.9800802</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="56303" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1887">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:44Z</infon>
<location offset="56358" length="8"/>
<text>subunits</text>
</annotation>
<annotation id="1347">
<infon key="score">0.998933</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56374" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1348">
<infon key="score">0.9990646</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="56385" length="4"/>
<text>iron</text>
</annotation>
<annotation id="2035">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:14Z</infon>
<location offset="56391" length="12"/>
<text>Coordination</text>
</annotation>
<annotation id="1349">
<infon key="score">0.998568</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:54:58Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="56407" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1350">
<infon key="score">0.9987746</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56440" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="1351">
<infon key="score">0.9989641</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:58:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56468" length="6"/>
<text>active</text>
</annotation>
<annotation id="1352">
<infon key="score">0.57369035</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56475" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1353">
<infon key="score">0.99948394</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56509" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="1354">
<infon key="score">0.9994797</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56519" length="5"/>
<text>Glu62</text>
</annotation>
<annotation id="1355">
<infon key="score">0.90486306</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56532" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1356">
<infon key="score">0.9986803</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56630" length="8"/>
<text>decamers</text>
</annotation>
<annotation id="1357">
<infon key="score">0.9472394</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56691" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1358">
<infon key="score">0.999086</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="56702" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1359">
<infon key="score">0.9983797</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56707" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="2094">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:30:46Z</infon>
<location offset="56742" length="7"/>
<text>loss of</text>
</annotation>
<annotation id="1360">
<infon key="score">0.9975012</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:13:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="56755" length="26"/>
<text>metal coordinating residue</text>
</annotation>
<annotation id="1361">
<infon key="score">0.99875724</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="56839" length="7"/>
<text>monomer</text>
</annotation>
</passage>
<passage>
<infon key="file">tbl4.xml</infon>
<infon key="id">tbl4</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>56879</offset>
<text>Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses. Friedel mates were averaged when calculating reflection numbers and statistics.</text>
<annotation id="2136">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:58:53Z</infon>
<location offset="56879" length="41"/>
<text>Data collection and refinement statistics</text>
</annotation>
</passage>
<passage>
<infon key="file">tbl4.xml</infon>
<infon key="id">tbl4</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>57072</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.025</text>
</passage>
<passage>
<infon key="file">tbl4.xml</infon>
<infon key="id">tbl4</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table</infon>
<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"/><th rowspan="1" colspan="1"><styled-content style="color: #000000;">WT</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">E32A</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">E62A</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">H65A</styled-content></th></tr></thead><tbody><tr><td rowspan="1" colspan="1">Data collection</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">Wavelength (Å)</td><td rowspan="1" colspan="1">1.74</td><td rowspan="1" colspan="1">1.73</td><td rowspan="1" colspan="1">1.73</td><td rowspan="1" colspan="1">1.74</td></tr><tr><td rowspan="1" colspan="1">Resolution range (Å)</td><td rowspan="1" colspan="1">49.63 - 2.06 <break/>(2.10 - 2.06)</td><td rowspan="1" colspan="1">48.84 - 2.59 <break/>(2.683 - 2.59)</td><td rowspan="1" colspan="1">48.87 - 2.21 <break/>(2.29 - 2.21)</td><td rowspan="1" colspan="1">48.86 - 2.97 <break/>(3.08 - 2.97)</td></tr><tr><td rowspan="1" colspan="1">Space group</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td></tr><tr><td rowspan="1" colspan="1">Unit cell (Å) <italic>a</italic>
<break/> b <break/>
<italic> c</italic>
<break/> β (°)</td><td rowspan="1" colspan="1">98.18 <break/>120.53 <break/>140.30 <break/>95.36</td><td rowspan="1" colspan="1">97.78 <break/>120.28 <break/>140.53 <break/>95.41</td><td rowspan="1" colspan="1">98.09 <break/>120.23 <break/>140.36 <break/>95.50</td><td rowspan="1" colspan="1">98.03 <break/>120.29 <break/>140.43 <break/>95.39</td></tr><tr><td rowspan="1" colspan="1">Total reflections</td><td rowspan="1" colspan="1">1,264,922 <break/>(41,360)</td><td rowspan="1" colspan="1">405,488 <break/>(36,186)</td><td rowspan="1" colspan="1">1,069,345 <break/>(95,716)</td><td rowspan="1" colspan="1">323,853 <break/>(32,120)</td></tr><tr><td rowspan="1" colspan="1">Unique reflections</td><td rowspan="1" colspan="1">197,873 <break/>(8,766)</td><td rowspan="1" colspan="1">100,067 <break/>(9,735)</td><td rowspan="1" colspan="1">162,379 <break/>(15,817)</td><td rowspan="1" colspan="1">66,658 <break/>(6,553)</td></tr><tr><td rowspan="1" colspan="1">Multiplicity</td><td rowspan="1" colspan="1">6.4 (4.7)</td><td rowspan="1" colspan="1">4.1 (3.7)</td><td rowspan="1" colspan="1">6.6 (6.1)</td><td rowspan="1" colspan="1">4.9 (4.9)</td></tr><tr><td rowspan="1" colspan="1">Anomalous multiplicity</td><td rowspan="1" colspan="1">3.2 (2.6)</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td></tr><tr><td rowspan="1" colspan="1">Completeness (%)</td><td rowspan="1" colspan="1">99.2 (88.6)</td><td rowspan="1" colspan="1">99.0 (97.0)</td><td rowspan="1" colspan="1">100 (97.0)</td><td rowspan="1" colspan="1">100 (99.0)</td></tr><tr><td rowspan="1" colspan="1">Anomalous completeness (%)</td><td rowspan="1" colspan="1">96.7 (77.2)</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td></tr><tr><td rowspan="1" colspan="1">Mean I/sigma(I)</td><td rowspan="1" colspan="1">10.6 (1.60)</td><td rowspan="1" colspan="1">8.46 (1.79)</td><td rowspan="1" colspan="1">13.74 (1.80)</td><td rowspan="1" colspan="1">8.09 (1.74)</td></tr><tr><td rowspan="1" colspan="1">Wilson B-factor</td><td rowspan="1" colspan="1">26.98</td><td rowspan="1" colspan="1">40.10</td><td rowspan="1" colspan="1">33.97</td><td rowspan="1" colspan="1">52.20</td></tr><tr><td rowspan="1" colspan="1">R<sub>merge</sub></td><td rowspan="1" colspan="1">0.123 (0.790)</td><td rowspan="1" colspan="1">0.171 (0.792)</td><td rowspan="1" colspan="1">0.0979 (1.009)</td><td rowspan="1" colspan="1">0.177 (0.863)</td></tr><tr><td rowspan="1" colspan="1">R<sub>meas</sub></td><td rowspan="1" colspan="1">0.147 (0.973)</td><td rowspan="1" colspan="1">0.196 (0.923)</td><td rowspan="1" colspan="1">0.1064 (1.107)</td><td rowspan="1" colspan="1">0.199 (0.966)</td></tr><tr><td rowspan="1" colspan="1">CC1/2</td><td rowspan="1" colspan="1">0.995 (0.469)</td><td rowspan="1" colspan="1">0.985 (0.557)</td><td rowspan="1" colspan="1">0.998 (0.642)</td><td rowspan="1" colspan="1">0.989 (0.627)</td></tr><tr><td rowspan="1" colspan="1">CC*</td><td rowspan="1" colspan="1">0.999 (0.846)</td><td rowspan="1" colspan="1">0.996 (0.846)</td><td rowspan="1" colspan="1">0.999 (0.884)</td><td rowspan="1" colspan="1">0.997 (0.878)</td></tr><tr><td rowspan="1" colspan="1">Image DOI</td><td rowspan="1" colspan="1">10.7488/ds/1342</td><td rowspan="1" colspan="1">10.7488/ds/1419</td><td rowspan="1" colspan="1">10.7488/ds/1420</td><td rowspan="1" colspan="1">10.7488/ds/1421</td></tr><tr><td rowspan="1" colspan="1">Refinement</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">R<sub>work</sub></td><td rowspan="1" colspan="1">0.171 (0.318)</td><td rowspan="1" colspan="1">0.183 (0.288)</td><td rowspan="1" colspan="1">0.165 (0.299)</td><td rowspan="1" colspan="1">0.186 (0.273)</td></tr><tr><td rowspan="1" colspan="1">R<sub>free</sub></td><td rowspan="1" colspan="1">0.206 (0.345)</td><td rowspan="1" colspan="1">0.225 (0351)</td><td rowspan="1" colspan="1">0.216 (0.364)</td><td rowspan="1" colspan="1">0.237 (0.325)</td></tr><tr><td rowspan="1" colspan="1">Number of non-hydrogen atoms</td><td rowspan="1" colspan="1">23,222</td><td rowspan="1" colspan="1">22,366</td><td rowspan="1" colspan="1">22,691</td><td rowspan="1" colspan="1">22,145</td></tr><tr><td rowspan="1" colspan="1">macromolecules</td><td rowspan="1" colspan="1">22,276</td><td rowspan="1" colspan="1">22,019</td><td rowspan="1" colspan="1">21,965</td><td rowspan="1" colspan="1">22,066</td></tr><tr><td rowspan="1" colspan="1">ligands</td><td rowspan="1" colspan="1">138</td><td rowspan="1" colspan="1">8</td><td rowspan="1" colspan="1">24</td><td rowspan="1" colspan="1">74</td></tr><tr><td rowspan="1" colspan="1">water</td><td rowspan="1" colspan="1">808</td><td rowspan="1" colspan="1">339</td><td rowspan="1" colspan="1">702</td><td rowspan="1" colspan="1">5</td></tr><tr><td rowspan="1" colspan="1">Protein residues</td><td rowspan="1" colspan="1">2,703</td><td rowspan="1" colspan="1">2,686</td><td rowspan="1" colspan="1">2,675</td><td rowspan="1" colspan="1">2,700</td></tr><tr><td rowspan="1" colspan="1">RMS(bonds) (Å)</td><td rowspan="1" colspan="1">0.012</td><td rowspan="1" colspan="1">0.005</td><td rowspan="1" colspan="1">0.011</td><td rowspan="1" colspan="1">0.002</td></tr><tr><td rowspan="1" colspan="1">RMS(angles) (°)</td><td rowspan="1" colspan="1">1.26</td><td rowspan="1" colspan="1">0.58</td><td rowspan="1" colspan="1">1.02</td><td rowspan="1" colspan="1">0.40</td></tr><tr><td rowspan="1" colspan="1">Ramachandran favored (%)</td><td rowspan="1" colspan="1">100</td><td rowspan="1" colspan="1">99</td><td rowspan="1" colspan="1">100</td><td rowspan="1" colspan="1">99</td></tr><tr><td rowspan="1" colspan="1">Ramachandran allowed (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1">Ramachandran outliers (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Clash score</td><td rowspan="1" colspan="1">1.42</td><td rowspan="1" colspan="1">1.42</td><td rowspan="1" colspan="1">1.79</td><td rowspan="1" colspan="1">0.97</td></tr><tr><td rowspan="1" colspan="1">Average B-factor (Å<sup>2</sup>)</td><td rowspan="1" colspan="1">33.90</td><td rowspan="1" colspan="1">42.31</td><td rowspan="1" colspan="1">41.34</td><td rowspan="1" colspan="1">47.68</td></tr><tr><td rowspan="1" colspan="1">macromolecules</td><td rowspan="1" colspan="1">33.80</td><td rowspan="1" colspan="1">42.35</td><td rowspan="1" colspan="1">41.31</td><td rowspan="1" colspan="1">47.60</td></tr><tr><td rowspan="1" colspan="1">ligands</td><td rowspan="1" colspan="1">40.40</td><td rowspan="1" colspan="1">72.80</td><td rowspan="1" colspan="1">65.55</td><td rowspan="1" colspan="1">72.34</td></tr><tr><td rowspan="1" colspan="1">solvent</td><td rowspan="1" colspan="1">36.20</td><td rowspan="1" colspan="1">38.95</td><td rowspan="1" colspan="1">41.46</td><td rowspan="1" colspan="1">33.85</td></tr><tr><td rowspan="1" colspan="1">PDB ID</td><td rowspan="1" colspan="1">5DA5</td><td rowspan="1" colspan="1">5L89</td><td rowspan="1" colspan="1">5L8B</td><td rowspan="1" colspan="1">5L8G</td></tr></tbody></table>
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<offset>57119</offset>
<text> WT E32A E62A H65A Data collection Wavelength (Å) 1.74 1.73 1.73 1.74 Resolution range (Å) 49.63 - 2.06 (2.10 - 2.06) 48.84 - 2.59 (2.683 - 2.59) 48.87 - 2.21 (2.29 - 2.21) 48.86 - 2.97 (3.08 - 2.97) Space group P 1 21 1 P 1 21 1 P 1 21 1 P 1 21 1 Unit cell (Å) a b c β (°) 98.18 120.53 140.30 95.36 97.78 120.28 140.53 95.41 98.09 120.23 140.36 95.50 98.03 120.29 140.43 95.39 Total reflections 1,264,922 (41,360) 405,488 (36,186) 1,069,345 (95,716) 323,853 (32,120) Unique reflections 197,873 (8,766) 100,067 (9,735) 162,379 (15,817) 66,658 (6,553) Multiplicity 6.4 (4.7) 4.1 (3.7) 6.6 (6.1) 4.9 (4.9) Anomalous multiplicity 3.2 (2.6) N/A N/A N/A Completeness (%) 99.2 (88.6) 99.0 (97.0) 100 (97.0) 100 (99.0) Anomalous completeness (%) 96.7 (77.2) N/A N/A N/A Mean I/sigma(I) 10.6 (1.60) 8.46 (1.79) 13.74 (1.80) 8.09 (1.74) Wilson B-factor 26.98 40.10 33.97 52.20 Rmerge 0.123 (0.790) 0.171 (0.792) 0.0979 (1.009) 0.177 (0.863) Rmeas 0.147 (0.973) 0.196 (0.923) 0.1064 (1.107) 0.199 (0.966) CC1/2 0.995 (0.469) 0.985 (0.557) 0.998 (0.642) 0.989 (0.627) CC* 0.999 (0.846) 0.996 (0.846) 0.999 (0.884) 0.997 (0.878) Image DOI 10.7488/ds/1342 10.7488/ds/1419 10.7488/ds/1420 10.7488/ds/1421 Refinement Rwork 0.171 (0.318) 0.183 (0.288) 0.165 (0.299) 0.186 (0.273) Rfree 0.206 (0.345) 0.225 (0351) 0.216 (0.364) 0.237 (0.325) Number of non-hydrogen atoms 23,222 22,366 22,691 22,145 macromolecules 22,276 22,019 21,965 22,066 ligands 138 8 24 74 water 808 339 702 5 Protein residues 2,703 2,686 2,675 2,700 RMS(bonds) (Å) 0.012 0.005 0.011 0.002 RMS(angles) (°) 1.26 0.58 1.02 0.40 Ramachandran favored (%) 100 99 100 99 Ramachandran allowed (%) 0 1 0 1 Ramachandran outliers (%) 0 0 0 0 Clash score 1.42 1.42 1.79 0.97 Average B-factor (Å2) 33.90 42.31 41.34 47.68 macromolecules 33.80 42.35 41.31 47.60 ligands 40.40 72.80 65.55 72.34 solvent 36.20 38.95 41.46 33.85 PDB ID 5DA5 5L89 5L8B 5L8G </text>
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<text>WT</text>
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<text>Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation.</text>
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<text>Iron</text>
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<text>EncFtn</text>
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<text>encapsulin</text>
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<infon key="updated_at">2023-09-19T11:28:05Z</infon>
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<text>ferritin</text>
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<infon key="score">0.9986656</infon>
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<infon key="updated_at">2023-09-19T11:32:19Z</infon>
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<location offset="59207" length="9"/>
<text>decameric</text>
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<infon key="score">0.9990393</infon>
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<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="59217" length="8"/>
<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="59227" length="10"/>
<text>encapsulin</text>
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<infon key="score">0.9954901</infon>
<infon key="type">complex_assembly</infon>
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<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="59239" length="10"/>
<text>EncFtn-Enc</text>
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<infon key="score">0.9988637</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="59254" length="11"/>
<text>apoferritin</text>
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<infon key="score">0.998739</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="59282" length="13"/>
<text>Fe(NH4)2(SO4)</text>
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<infon key="score">0.53595287</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:35Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>HCl</text>
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<infon key="updated_at">2023-09-19T14:59:31Z</infon>
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<text>NaCl</text>
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<infon key="score">0.9981086</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="59441" length="2"/>
<text>Fe</text>
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<infon key="score">0.99486285</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:38Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>Fe</text>
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<infon key="score">0.99608636</infon>
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<infon key="updated_at">2023-09-19T15:00:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="59577" length="15"/>
<text>ferrozine assay</text>
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<infon key="score">0.9982328</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:00:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="59597" length="20"/>
<text>BCA microplate assay</text>
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<infon key="score">0.997769</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="59633" length="2"/>
<text>Fe</text>
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<infon key="score">0.99900657</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:40Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>Fe</text>
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<infon key="score">0.99914956</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>iron</text>
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<infon key="score">0.999042</infon>
<infon key="type">chemical</infon>
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<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>iron</text>
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<infon key="score">0.9990244</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="59887" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1387">
<infon key="score">0.99873203</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60008" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1388">
<infon key="score">0.99927837</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60020" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1389">
<infon key="score">0.9973688</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:00:15Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>60mer</text>
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<infon key="score">0.9991992</infon>
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<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
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<text>encapsulin</text>
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<infon key="score">0.99671483</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60056" length="5"/>
<text>60mer</text>
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<infon key="score">0.9990583</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60065" length="10"/>
<text>encapsulin</text>
</annotation>
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<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:39:43Z</infon>
<location offset="60076" length="11"/>
<text>loaded with</text>
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<infon key="score">0.99869376</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60101" length="9"/>
<text>decameric</text>
</annotation>
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<infon key="score">0.99931586</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60111" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1395">
<infon key="score">0.9948677</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:00:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60138" length="5"/>
<text>24mer</text>
</annotation>
<annotation id="1396">
<infon key="score">0.9613902</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:00:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60148" length="5"/>
<text>horse</text>
</annotation>
<annotation id="1397">
<infon key="score">0.9992094</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60161" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="2137">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:00:49Z</infon>
<location offset="60257" length="35"/>
<text>ferrozine and BCA microplate assays</text>
</annotation>
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<infon key="score">0.99502254</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:59:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="60315" length="2"/>
<text>Fe</text>
</annotation>
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<infon key="file">tbl5.xml</infon>
<infon key="id">tbl5</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>60373</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.026</text>
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<passage>
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<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Protein sample</th><th rowspan="1" colspan="1">Fe(NH<sub>4)2</sub>(SO<sub>4)2</sub> loading (µM)</th><th rowspan="1" colspan="1">Fe detected by ferrozine assay (µM)</th><th rowspan="1" colspan="1">Protein detected by BCA microplate assay (µM)</th><th rowspan="1" colspan="1">Fe / monomeric protein</th><th rowspan="1" colspan="1">Maximum Fe loading per biological assembly unit</th></tr></thead><tbody><tr><td rowspan="6" colspan="1">8.46 µM EncFtn<sub>sH</sub>-10mer</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">4.73 ± 2.32</td><td rowspan="1" colspan="1">5.26 ± 0.64</td><td rowspan="1" colspan="1">0.90 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">39.9</td><td rowspan="1" colspan="1">9.93 ± 1.20</td><td rowspan="1" colspan="1">5.36 ± 0.69</td><td rowspan="1" colspan="1">1.85 ± 0.22</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">84</td><td rowspan="1" colspan="1">17.99 ± 2.01</td><td rowspan="1" colspan="1">4.96 ± 0.04</td><td rowspan="1" colspan="1">3.63 ± 0.41</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">147</td><td rowspan="1" colspan="1">21.09 ± 1.94</td><td rowspan="1" colspan="1">4.44 ± 0.21</td><td rowspan="1" colspan="1">4.75 ± 0.44</td><td rowspan="1" colspan="1">48 ± 4</td></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">28.68 ± 0.30</td><td rowspan="1" colspan="1">3.73 ± 0.53</td><td rowspan="1" colspan="1">7.68 ± 0.08</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">11.27 ± 1.10</td><td rowspan="1" colspan="1">2.50 ± 0.05</td><td rowspan="1" colspan="1">4.51 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="8" colspan="1">8.50 µM Encapsulin</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">-1.02 ± 0.54</td><td rowspan="1" colspan="1">8.63 ± 0.17</td><td rowspan="1" colspan="1">-0.12 ± 0.06</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">62.24 ± 2.49</td><td rowspan="1" colspan="1">10.01 ± 0.58</td><td rowspan="1" colspan="1">6.22 ± 0.35</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">67.94 ± 3.15</td><td rowspan="1" colspan="1">8.69 ± 0.42</td><td rowspan="1" colspan="1">7.81 ± 0.36</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">450</td><td rowspan="1" colspan="1">107.96 ± 8.88</td><td rowspan="1" colspan="1">8.50 ± 0.69</td><td rowspan="1" colspan="1">12.71 ± 1.05</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">700</td><td rowspan="1" colspan="1">97.51 ± 3.19</td><td rowspan="1" colspan="1">7.26 ± 0.20</td><td rowspan="1" colspan="1">13.44 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1000</td><td rowspan="1" colspan="1">308.63 ± 2.06</td><td rowspan="1" colspan="1">8.42 ± 0.34</td><td rowspan="1" colspan="1">36.66 ± 0.24</td><td rowspan="1" colspan="1">2199 ± 15</td></tr><tr><td rowspan="1" colspan="1">1500</td><td rowspan="1" colspan="1">57.09 ± 0.90</td><td rowspan="1" colspan="1">1.44 ± 0.21</td><td rowspan="1" colspan="1">39.77 ± 0.62</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">2000</td><td rowspan="1" colspan="1">9.2 ± 1.16</td><td rowspan="1" colspan="1">0.21 ± 0.14</td><td rowspan="1" colspan="1">44.73 ± 5.63</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="8" colspan="1">8.70 µM EncFtn-Enc</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">3.31 ± 1.57</td><td rowspan="1" colspan="1">6.85 ± 0.07</td><td rowspan="1" colspan="1">0.48 ± 0.23</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">116.27 ± 3.74</td><td rowspan="1" colspan="1">7.63 ± 0.12</td><td rowspan="1" colspan="1">15.25 ± 0.49</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">132.86 ± 4.03</td><td rowspan="1" colspan="1">6.66 ± 0.31</td><td rowspan="1" colspan="1">19.96 ± 0.61</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">450</td><td rowspan="1" colspan="1">220.57 ± 27.33</td><td rowspan="1" colspan="1">6.12 ± 1.07</td><td rowspan="1" colspan="1">36.06 ± 4.47</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">700</td><td rowspan="1" colspan="1">344.03 ± 40.38</td><td rowspan="1" colspan="1">6.94 ± 0.17</td><td rowspan="1" colspan="1">49.58 ± 5.82</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1000</td><td rowspan="1" colspan="1">496.00 ± 38.48</td><td rowspan="1" colspan="1">7.19 ± 0.08</td><td rowspan="1" colspan="1">68.94 ± 5.35</td><td rowspan="1" colspan="1">4137 ± 321</td></tr><tr><td rowspan="1" colspan="1">1500</td><td rowspan="1" colspan="1">569.98 ± 73.63</td><td rowspan="1" colspan="1">5.73 ± 0.03</td><td rowspan="1" colspan="1">99.44 ± 12.84</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">2000</td><td rowspan="1" colspan="1">584.30 ± 28.33</td><td rowspan="1" colspan="1">4.88 ± 0.22</td><td rowspan="1" colspan="1">119.62 ± 5.80</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="6" colspan="1">8.50 µM Apoferritin</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">3.95 ± 2.26</td><td rowspan="1" colspan="1">9.37 ± 0.24</td><td rowspan="1" colspan="1">0.42 ± 0.25</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">42.5</td><td rowspan="1" colspan="1">10.27 ± 1.12</td><td rowspan="1" colspan="1">8.27 ± 0.30</td><td rowspan="1" colspan="1">1.24 ± 0.18</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">212.5</td><td rowspan="1" colspan="1">44.48 ± 2.76</td><td rowspan="1" colspan="1">7.85 ± 0.77</td><td rowspan="1" colspan="1">5.67 ± 0.83</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">637.5</td><td rowspan="1" colspan="1">160.93 ± 4.27</td><td rowspan="1" colspan="1">6.76 ± 0.81</td><td rowspan="1" colspan="1">23.79 ± 3.12</td><td rowspan="1" colspan="1">571 ± 75</td></tr><tr><td rowspan="1" colspan="1">1275</td><td rowspan="1" colspan="1">114.92 ± 3.17</td><td rowspan="1" colspan="1">3.84 ± 0.30</td><td rowspan="1" colspan="1">29.91 ± 2.95</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1700</td><td rowspan="1" colspan="1">91.40 ± 3.37</td><td rowspan="1" colspan="1">3.14 ± 0.35</td><td rowspan="1" colspan="1">29.13 ± 3.86</td><td rowspan="1" colspan="1"/></tr></tbody></table>
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<text>Protein sample Fe(NH4)2(SO4)2 loading (µM) Fe detected by ferrozine assay (µM) Protein detected by BCA microplate assay (µM) Fe / monomeric protein Maximum Fe loading per biological assembly unit 8.46 µM EncFtnsH-10mer 0 4.73 ± 2.32 5.26 ± 0.64 0.90 ± 0.44 39.9 9.93 ± 1.20 5.36 ± 0.69 1.85 ± 0.22 84 17.99 ± 2.01 4.96 ± 0.04 3.63 ± 0.41 147 21.09 ± 1.94 4.44 ± 0.21 4.75 ± 0.44 48 ± 4 224 28.68 ± 0.30 3.73 ± 0.53 7.68 ± 0.08 301 11.27 ± 1.10 2.50 ± 0.05 4.51 ± 0.44 8.50 µM Encapsulin 0 -1.02 ± 0.54 8.63 ± 0.17 -0.12 ± 0.06 224 62.24 ± 2.49 10.01 ± 0.58 6.22 ± 0.35 301 67.94 ± 3.15 8.69 ± 0.42 7.81 ± 0.36 450 107.96 ± 8.88 8.50 ± 0.69 12.71 ± 1.05 700 97.51 ± 3.19 7.26 ± 0.20 13.44 ± 0.44 1000 308.63 ± 2.06 8.42 ± 0.34 36.66 ± 0.24 2199 ± 15 1500 57.09 ± 0.90 1.44 ± 0.21 39.77 ± 0.62 2000 9.2 ± 1.16 0.21 ± 0.14 44.73 ± 5.63 8.70 µM EncFtn-Enc 0 3.31 ± 1.57 6.85 ± 0.07 0.48 ± 0.23 224 116.27 ± 3.74 7.63 ± 0.12 15.25 ± 0.49 301 132.86 ± 4.03 6.66 ± 0.31 19.96 ± 0.61 450 220.57 ± 27.33 6.12 ± 1.07 36.06 ± 4.47 700 344.03 ± 40.38 6.94 ± 0.17 49.58 ± 5.82 1000 496.00 ± 38.48 7.19 ± 0.08 68.94 ± 5.35 4137 ± 321 1500 569.98 ± 73.63 5.73 ± 0.03 99.44 ± 12.84 2000 584.30 ± 28.33 4.88 ± 0.22 119.62 ± 5.80 8.50 µM Apoferritin 0 3.95 ± 2.26 9.37 ± 0.24 0.42 ± 0.25 42.5 10.27 ± 1.12 8.27 ± 0.30 1.24 ± 0.18 212.5 44.48 ± 2.76 7.85 ± 0.77 5.67 ± 0.83 637.5 160.93 ± 4.27 6.76 ± 0.81 23.79 ± 3.12 571 ± 75 1275 114.92 ± 3.17 3.84 ± 0.30 29.91 ± 2.95 1700 91.40 ± 3.37 3.14 ± 0.35 29.13 ± 3.86 </text>
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<text>To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics). The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure. All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.</text>
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<text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. (B) Views of the FOC of the EncFtnsH-E32Amutant. Protein atoms shown as in (A).</text>
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http://dx.doi.org/10.7554/eLife.18972.028</text>
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<text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant. Protein atoms shown as in (A).</text>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.029</text>
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<passage>
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<infon key="section_type">FIG</infon>
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<text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines. (B) Views of the FOC of the EncFtnsH-H65A mutant. Protein atoms and metal ions shown as in (A).</text>
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<text>EncFtnsH-H65A</text>
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<location offset="63791" length="29"/>
<text>2mFo-DFc electron density map</text>
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<text>calcium</text>
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<infon key="updated_at">2023-09-19T11:54:10Z</infon>
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<location offset="64027" length="13"/>
<text>EncFtnsH-H65A</text>
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<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.030</text>
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<passage>
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<infon key="id">fig11</infon>
<infon key="section_type">FIG</infon>
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<offset>64146</offset>
<text>Comparison of the EncFtnsH FOC mutants vs wild type.</text>
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<text>EncFtnsH</text>
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<text>FOC</text>
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<infon key="updated_at">2023-09-19T14:55:40Z</infon>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>64199</offset>
<text>The structures of the three EncFtnsH mutants were all determined by X-ray crystallography. The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type. EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions. The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue. Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition. The overall organization of FOC residues is retained in the mutants, with almost no backbone movements. Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.</text>
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<text>X-ray crystallography</text>
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<infon key="updated_at">2023-09-19T14:53:14Z</infon>
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<infon key="updated_at">2023-09-19T14:53:21Z</infon>
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<infon key="updated_at">2023-09-19T14:53:26Z</infon>
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<infon key="updated_at">2023-09-19T14:55:40Z</infon>
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<infon key="updated_at">2023-09-19T15:05:49Z</infon>
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<text>crystallized</text>
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<infon key="updated_at">2023-09-19T15:05:41Z</infon>
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<infon key="updated_at">2023-09-19T13:14:01Z</infon>
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<location offset="64382" length="8"/>
<text>EncFtnsH</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:05:55Z</infon>
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<text>structure</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64478" length="3"/>
<text>FOC</text>
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<infon key="updated_at">2023-09-19T14:55:40Z</infon>
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<text>mutants</text>
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<infon key="updated_at">2023-09-19T16:22:15Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>native</text>
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<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
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<text>EncFtnsH</text>
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<infon key="updated_at">2023-09-19T15:05:58Z</infon>
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<text>structures</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:06:45Z</infon>
<location offset="64562" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="1467">
<infon key="score">0.9991374</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:55:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="64574" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1468">
<infon key="score">0.9991269</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:55:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="64593" length="4"/>
<text>Ca2+</text>
</annotation>
<annotation id="1469">
<infon key="score">0.9990487</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:05:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64642" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="1470">
<infon key="score">0.998978</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64659" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1471">
<infon key="score">0.99890566</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64671" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1472">
<infon key="score">0.998872</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64684" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="2129">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="64703" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1473">
<infon key="score">0.9981457</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64717" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="2143">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:06:30Z</infon>
<location offset="64730" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="1474">
<infon key="score">0.9984199</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="64773" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="1475">
<infon key="score">0.820491</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64850" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1476">
<infon key="score">0.6792209</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64882" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1477">
<infon key="score">0.9995347</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64964" length="5"/>
<text>Tyr39</text>
</annotation>
<annotation id="2145">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:07:03Z</infon>
<location offset="64986" length="10"/>
<text>coordinate</text>
</annotation>
<annotation id="2146">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:07:12Z</infon>
<location offset="65001" length="5"/>
<text>bound</text>
</annotation>
<annotation id="1478">
<infon key="score">0.9985001</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="65007" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="1479">
<infon key="score">0.9988858</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="65027" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1480">
<infon key="score">0.97360635</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65032" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1481">
<infon key="score">0.99951637</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65044" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="1482">
<infon key="score">0.99818367</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:06:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65096" length="9"/>
<text>structure</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig11.jpg</infon>
<infon key="id">fig11</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>65108</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.027</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>65155</offset>
<text>Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3). In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site. The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1). The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2). The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC. E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC. Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3). A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.</text>
<annotation id="1483">
<infon key="score">0.99103326</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65212" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="2130">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<location offset="65231" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1484">
<infon key="score">0.9991116</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="65336" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1485">
<infon key="score">0.98977494</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65341" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1486">
<infon key="score">0.9979477</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:07:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65397" length="26"/>
<text>iron coordinating residues</text>
</annotation>
<annotation id="1487">
<infon key="score">0.9932214</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65431" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1488">
<infon key="score">0.9947363</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65469" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="2147">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:08:16Z</infon>
<location offset="65496" length="12"/>
<text>coordinating</text>
</annotation>
<annotation id="1489">
<infon key="score">0.9994023</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65532" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="2148">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:09:25Z</infon>
<location offset="65549" length="23"/>
<text>abrogates metal binding</text>
</annotation>
<annotation id="1490">
<infon key="score">0.993806</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:13:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65591" length="13"/>
<text>Glu31/34-site</text>
</annotation>
<annotation id="1491">
<infon key="score">0.9983423</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:08:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65610" length="5"/>
<text>lacks</text>
</annotation>
<annotation id="1492">
<infon key="score">0.5333656</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:55:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="65616" length="5"/>
<text>metal</text>
</annotation>
<annotation id="1493">
<infon key="score">0.99938786</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65646" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="1494">
<infon key="score">0.9989635</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65685" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1495">
<infon key="score">0.82604885</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:55:18Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="65696" length="5"/>
<text>metal</text>
</annotation>
<annotation id="1496">
<infon key="score">0.9990553</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="65739" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1497">
<infon key="score">0.9840895</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65744" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1498">
<infon key="score">0.96367645</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65779" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1499">
<infon key="score">0.9990897</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="65790" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1500">
<infon key="score">0.9948501</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65795" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1501">
<infon key="score">0.99791396</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:14:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65815" length="10"/>
<text>entry site</text>
</annotation>
<annotation id="1502">
<infon key="score">0.9988519</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="65838" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="2152">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:10:22Z</infon>
<location offset="65850" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="1503">
<infon key="score">0.99943465</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65879" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="1504">
<infon key="score">0.99941957</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65889" length="5"/>
<text>Glu34</text>
</annotation>
<annotation id="1505">
<infon key="score">0.99890864</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="65932" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1506">
<infon key="score">0.99673647</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65937" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="1507">
<infon key="score">0.9989923</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:05:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65976" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="1508">
<infon key="score">0.9994692</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66018" length="5"/>
<text>Glu32</text>
</annotation>
<annotation id="1509">
<infon key="score">0.9995042</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66028" length="5"/>
<text>Tyr39</text>
</annotation>
<annotation id="1510">
<infon key="score">0.92200243</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="66041" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1511">
<infon key="score">0.9995846</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:23:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66046" length="3"/>
<text>E32</text>
</annotation>
<annotation id="1512">
<infon key="score">0.9989628</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:05:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66100" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="2150">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:09:53Z</infon>
<location offset="66114" length="11"/>
<text>coordinates</text>
</annotation>
<annotation id="1513">
<infon key="score">0.9990132</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:55:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="66126" length="4"/>
<text>Ca2+</text>
</annotation>
<annotation id="2149">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:09:42Z</infon>
<location offset="66206" length="11"/>
<text>coordinated</text>
</annotation>
<annotation id="1514">
<infon key="score">0.99841416</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="66218" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="1515">
<infon key="score">0.9188203</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="66237" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1516">
<infon key="score">0.99954957</infon>
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<location offset="66242" length="5"/>
<text>Tyr39</text>
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<infon key="identifier">CHEBI:</infon>
<location offset="66264" length="4"/>
<text>Ca2+</text>
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<infon key="updated_at">2023-09-19T14:55:48Z</infon>
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<text>wild-type</text>
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<text>coordinates</text>
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<location offset="66369" length="7"/>
<text>calcium</text>
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<infon key="updated_at">2023-09-19T18:14:08Z</infon>
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<text>entry site</text>
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<location offset="66435" length="5"/>
<text>Glu31</text>
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<passage>
<infon key="section_type">RESULTS</infon>
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<text>Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein. While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization. These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.</text>
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<text>FOC</text>
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<infon key="updated_at">2023-09-19T11:30:43Z</infon>
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<location offset="66618" length="6"/>
<text>EncFtn</text>
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<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66659" length="9"/>
<text>decameric</text>
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<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66720" length="9"/>
<text>decameric</text>
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<infon key="updated_at">2023-09-19T18:08:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66733" length="8"/>
<text>crystals</text>
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<infon key="updated_at">2023-09-19T14:55:40Z</infon>
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<infon key="updated_at">2023-09-19T17:55:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<location offset="66934" length="3"/>
<text>FOC</text>
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<annotation id="1532">
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<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="66976" length="6"/>
<text>EncFtn</text>
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<passage>
<infon key="file">elife-18972-fig12-figsupp1.jpg</infon>
<infon key="id">fig12s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>66992</offset>
<text>Progress curves recording ferroxidase activity of EncFtnsH mutants. 20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.</text>
<annotation id="1533">
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<infon key="updated_at">2023-09-19T15:11:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="66992" length="15"/>
<text>Progress curves</text>
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<annotation id="1872">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="67018" length="11"/>
<text>ferroxidase</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="67042" length="8"/>
<text>EncFtnsH</text>
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<annotation id="1535">
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67051" length="7"/>
<text>mutants</text>
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<annotation id="1536">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67066" length="9"/>
<text>wild-type</text>
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<annotation id="1537">
<infon key="score">0.9990854</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="67076" length="8"/>
<text>EncFtnsH</text>
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<annotation id="1538">
<infon key="score">0.9986676</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67086" length="4"/>
<text>E32A</text>
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<annotation id="1539">
<infon key="score">0.99891603</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67092" length="4"/>
<text>E62A</text>
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<annotation id="1540">
<infon key="score">0.99892336</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67101" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1541">
<infon key="score">0.7189546</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67106" length="7"/>
<text>mutants</text>
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<annotation id="1968">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:57Z</infon>
<location offset="67153" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
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<passage>
<infon key="file">elife-18972-fig12-figsupp1.jpg</infon>
<infon key="id">fig12s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>67195</offset>
<text>Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation. Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls. Assays were performed with three technical repeats. Error bars were showed in shadows behind each curves.</text>
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<infon key="score">0.99893546</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:10:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="67268" length="4"/>
<text>Fe3+</text>
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<infon key="score">0.9990836</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:11:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="67349" length="4"/>
<text>Fe2+</text>
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<passage>
<infon key="file">elife-18972-fig12-figsupp1.jpg</infon>
<infon key="id">fig12s1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>67495</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.032</text>
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<passage>
<infon key="file">elife-18972-fig12.jpg</infon>
<infon key="id">fig12</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>67542</offset>
<text>Relative ferroxidase activity of EncFtnsH mutants.</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="67551" length="11"/>
<text>ferroxidase</text>
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<infon key="score">0.87679505</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="67575" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2131">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="67584" length="7"/>
<text>mutants</text>
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<passage>
<infon key="file">elife-18972-fig12.jpg</infon>
<infon key="id">fig12</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>67593</offset>
<text>EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2. Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation. The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315. Three technical repeats were performed and the plotted error bars represent the calculated standard deviations. The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.</text>
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<infon key="score">0.47456783</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="67593" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1546">
<infon key="score">0.9963652</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67611" length="6"/>
<text>mutant</text>
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<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67624" length="4"/>
<text>E32A</text>
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<infon key="score">0.99874085</infon>
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<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67630" length="4"/>
<text>E62A</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="67639" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1550">
<infon key="score">0.9989173</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:17:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="67690" length="14"/>
<text>Fe(NH4)2(SO4)2</text>
</annotation>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="67706" length="11"/>
<text>Ferroxidase</text>
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<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67734" length="6"/>
<text>mutant</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="67764" length="34"/>
<text>measuring the absorbance at 315 nm</text>
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<infon key="score">0.99880314</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:11:28Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="67839" length="4"/>
<text>Fe3+</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="67868" length="11"/>
<text>ferroxidase</text>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="67892" length="7"/>
<text>mutants</text>
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<infon key="updated_at">2023-09-19T14:55:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="67950" length="9"/>
<text>wild-type</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:11:43Z</infon>
<location offset="67987" length="19"/>
<text>measurement of A315</text>
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<infon key="score">0.8451369</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68124" length="3"/>
<text>FOC</text>
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="68128" length="7"/>
<text>mutants</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="68151" length="11"/>
<text>ferroxidase</text>
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<annotation id="1556">
<infon key="score">0.9987086</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68203" length="4"/>
<text>E62A</text>
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<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:30Z</infon>
<location offset="68236" length="11"/>
<text>ferroxidase</text>
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<passage>
<infon key="file">elife-18972-fig12.jpg</infon>
<infon key="id">fig12</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>68266</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.031</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>68313</offset>
<text>To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before. Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12). Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.</text>
<annotation id="1557">
<infon key="score">0.9969042</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68344" length="11"/>
<text>mutagenesis</text>
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<annotation id="1558">
<infon key="score">0.9987772</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:07:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68363" length="26"/>
<text>iron coordinating residues</text>
</annotation>
<annotation id="1559">
<infon key="score">0.9993548</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="68428" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1560">
<infon key="score">0.9609241</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="68457" length="15"/>
<text>progress curves</text>
</annotation>
<annotation id="1561">
<infon key="score">0.99912536</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="68494" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1562">
<infon key="score">0.999081</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="68502" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1563">
<infon key="score">0.9333361</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:55:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="68524" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="1564">
<infon key="score">0.99793833</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68543" length="11"/>
<text>Mutagenesis</text>
</annotation>
<annotation id="1565">
<infon key="score">0.99888545</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68558" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1566">
<infon key="score">0.9989735</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68567" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1567">
<infon key="score">0.9993606</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="68596" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1568">
<infon key="score">0.9990294</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68627" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1569">
<infon key="score">0.9976634</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:51:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="68632" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="2095">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:30:46Z</infon>
<location offset="68693" length="7"/>
<text>loss of</text>
</annotation>
<annotation id="2039">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:11:14Z</infon>
<location offset="68714" length="12"/>
<text>coordination</text>
</annotation>
<annotation id="1570">
<infon key="score">0.99380463</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68752" length="22"/>
<text>di-nuclear iron center</text>
</annotation>
<annotation id="1571">
<infon key="score">0.50114024</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68782" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1572">
<infon key="score">0.9930937</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="68827" length="8"/>
<text>mutating</text>
</annotation>
<annotation id="1573">
<infon key="score">0.61401594</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68858" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1574">
<infon key="score">0.9987893</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:07:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="68893" length="26"/>
<text>iron coordinating residues</text>
</annotation>
<annotation id="1878">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:31Z</infon>
<location offset="68928" length="11"/>
<text>ferroxidase</text>
</annotation>
<annotation id="1575">
<infon key="score">0.99936014</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="68956" length="8"/>
<text>EncFtnsH</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>68975</offset>
<text>Discussion</text>
</passage>
<passage>
<infon key="file">elife-18972-fig13.jpg</infon>
<infon key="id">fig13</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>68986</offset>
<text>Phylogenetic tree of ferritin family proteins.</text>
<annotation id="1576">
<infon key="score">0.848318</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:08:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="68986" length="17"/>
<text>Phylogenetic tree</text>
</annotation>
<annotation id="1841">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="69007" length="8"/>
<text>ferritin</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig13.jpg</infon>
<infon key="id">fig13</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>69033</offset>
<text>The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the likely root of the tree is indicated by a red arrow. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2.5). The analysis involved 104 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 </text>
<annotation id="1577">
<infon key="score">0.998771</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:13:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="69062" length="23"/>
<text>Neighbor-Joining method</text>
</annotation>
<annotation id="1578">
<infon key="score">0.99878114</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:13:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="69095" length="39"/>
<text>step-wise amino acid sequence alignment</text>
</annotation>
<annotation id="1579">
<infon key="score">0.9988074</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:13:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="69142" length="19"/>
<text>four-helical bundle</text>
</annotation>
<annotation id="1842">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="69174" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1580">
<infon key="score">0.9776418</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:13:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="69426" length="22"/>
<text>evolutionary distances</text>
</annotation>
<annotation id="1581">
<infon key="score">0.89482874</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:13:30Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="69473" length="17"/>
<text>p-distance method</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig13.jpg</infon>
<infon key="id">fig13</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>69870</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.033</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>69917</offset>
<text>Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc). By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture. Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A). A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn). The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13). While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ). From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn. This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B). Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).</text>
<annotation id="1843">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="69953" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1582">
<infon key="score">0.9077177</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="69977" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1583">
<infon key="score">0.998769</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="70012" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="1853">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<location offset="70022" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1914">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<location offset="70033" length="16"/>
<text>nanocompartments</text>
</annotation>
<annotation id="1584">
<infon key="score">0.9978338</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:11:18Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70051" length="3"/>
<text>Enc</text>
</annotation>
<annotation id="1585">
<infon key="score">0.6781485</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70073" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1586">
<infon key="score">0.99800014</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:56:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70085" length="9"/>
<text>R. rubrum</text>
</annotation>
<annotation id="1587">
<infon key="score">0.9974673</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="70115" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1588">
<infon key="score">0.73214674</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70151" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1589">
<infon key="score">0.99798334</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="70158" length="8"/>
<text>decamers</text>
</annotation>
<annotation id="1902">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="70271" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1590">
<infon key="score">0.9990447</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70281" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1591">
<infon key="score">0.99843824</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:14:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="70296" length="28"/>
<text>four-helical bundle scaffold</text>
</annotation>
<annotation id="1592">
<infon key="score">0.9819665</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="70329" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1593">
<infon key="score">0.8537571</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70336" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1594">
<infon key="score">0.9987708</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70371" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1595">
<infon key="score">0.9938612</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:14:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70395" length="32"/>
<text>sequence-based phylogenetic tree</text>
</annotation>
<annotation id="1844">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="70448" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1903">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="70500" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1596">
<infon key="score">0.9990343</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70510" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1597">
<infon key="score">0.9987172</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:14:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70521" length="17"/>
<text>bacterioferritins</text>
</annotation>
<annotation id="2156">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:14:52Z</infon>
<location offset="70543" length="3"/>
<text>Dps</text>
</annotation>
<annotation id="1598">
<infon key="score">0.9979739</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:15:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70583" length="44"/>
<text>encapsulin-associated ferritin-like proteins</text>
</annotation>
<annotation id="1599">
<infon key="score">0.99645376</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:15:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70629" length="7"/>
<text>EncFtns</text>
</annotation>
<annotation id="1600">
<infon key="score">0.93192923</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70684" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1601">
<infon key="score">0.7118862</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:15:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70705" length="10"/>
<text>Non-EncFtn</text>
</annotation>
<annotation id="1602">
<infon key="score">0.82859075</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="70749" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1603">
<infon key="score">0.6682153</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:15:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70760" length="10"/>
<text>Non-EncFtn</text>
</annotation>
<annotation id="1604">
<infon key="score">0.99898154</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="70845" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1605">
<infon key="score">0.9111064</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71002" length="20"/>
<text>active site scaffold</text>
</annotation>
<annotation id="1858">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:15:50Z</infon>
<location offset="71095" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1845">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="71136" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1606">
<infon key="score">0.9984102</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71243" length="17"/>
<text>four-helical fold</text>
</annotation>
<annotation id="1904">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="71268" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1607">
<infon key="score">0.99908197</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="71278" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1608">
<infon key="score">0.8914713</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="71347" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1609">
<infon key="score">0.75947845</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71397" length="17"/>
<text>C-terminal region</text>
</annotation>
<annotation id="1610">
<infon key="score">0.9945093</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="71422" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1611">
<infon key="score">0.9987987</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:13:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71429" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="1612">
<infon key="score">0.99855626</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71504" length="22"/>
<text>four-helix bundle fold</text>
</annotation>
<annotation id="1613">
<infon key="score">0.49689123</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71676" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1614">
<infon key="score">0.994453</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="71725" length="20"/>
<text>active-site residues</text>
</annotation>
<annotation id="1846">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<location offset="71762" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1615">
<infon key="score">0.9995757</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71799" length="5"/>
<text>Glu95</text>
</annotation>
<annotation id="1616">
<infon key="score">0.99958545</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71806" length="6"/>
<text>Gln128</text>
</annotation>
<annotation id="1617">
<infon key="score">0.99957746</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:16:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71817" length="6"/>
<text>Glu131</text>
</annotation>
<annotation id="1618">
<infon key="score">0.9937343</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:20:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="71827" length="5"/>
<text>PmFtn</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_2</infon>
<offset>71858</offset>
<text>Relationship between ferritin structure and activity</text>
<annotation id="1619">
<infon key="score">0.98743826</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="71879" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1620">
<infon key="score">0.55521035</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:08:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71888" length="9"/>
<text>structure</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>71911</offset>
<text>The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments. The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions. This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product. A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A). This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region). Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn. Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC. The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.</text>
<annotation id="1905">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="71941" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1621">
<infon key="score">0.99900573</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="71951" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1622">
<infon key="score">0.99534684</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="71969" length="10"/>
<text>octahedral</text>
</annotation>
<annotation id="1623">
<infon key="score">0.81837994</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="71980" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1624">
<infon key="score">0.96995956</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:12:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="71993" length="3"/>
<text>Dps</text>
</annotation>
<annotation id="1625">
<infon key="score">0.9974191</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:19:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="72004" length="9"/>
<text>dodecamer</text>
</annotation>
<annotation id="1626">
<infon key="score">0.9799951</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="72059" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1627">
<infon key="score">0.9988739</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="72122" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1628">
<infon key="score">0.99887925</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72297" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1629">
<infon key="score">0.9988193</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="72337" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1630">
<infon key="score">0.9952247</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:18:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="72418" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1631">
<infon key="score">0.9987408</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72449" length="20"/>
<text>structural alignment</text>
</annotation>
<annotation id="1632">
<infon key="score">0.9852278</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="72477" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1633">
<infon key="score">0.99501425</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="72484" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1906">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="72500" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1634">
<infon key="score">0.9988361</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72510" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1635">
<infon key="score">0.9954562</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:20:38Z</infon>
<infon key="identifier">PR:</infon>
<location offset="72519" length="5"/>
<text>PmFtn</text>
</annotation>
<annotation id="1636">
<infon key="score">0.99760485</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="72540" length="12"/>
<text>central ring</text>
</annotation>
<annotation id="1637">
<infon key="score">0.9990308</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="72556" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1638">
<infon key="score">0.99808574</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72602" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1639">
<infon key="score">0.9983908</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="72645" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1640">
<infon key="score">0.99829125</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:14:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="72680" length="22"/>
<text>mineralization surface</text>
</annotation>
<annotation id="1641">
<infon key="score">0.9971945</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72706" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1642">
<infon key="score">0.99885035</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:21:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72753" length="7"/>
<text>overlay</text>
</annotation>
<annotation id="1643">
<infon key="score">0.9989034</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:31:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="72790" length="18"/>
<text>ferroxidase center</text>
</annotation>
<annotation id="1644">
<infon key="score">0.9981205</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="72812" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1907">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="72867" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1645">
<infon key="score">0.99885356</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="72877" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1832">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<location offset="72927" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1646">
<infon key="score">0.8510018</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:22:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73024" length="9"/>
<text>mutations</text>
</annotation>
<annotation id="1647">
<infon key="score">0.9988943</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73035" length="4"/>
<text>E32A</text>
</annotation>
<annotation id="1648">
<infon key="score">0.998966</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73041" length="4"/>
<text>E62A</text>
</annotation>
<annotation id="1649">
<infon key="score">0.998933</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:53:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73050" length="4"/>
<text>H65A</text>
</annotation>
<annotation id="1650">
<infon key="score">0.8451171</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73068" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1651">
<infon key="score">0.9989415</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73105" length="26"/>
<text>iron-coordinating residues</text>
</annotation>
<annotation id="1652">
<infon key="score">0.9991235</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="73161" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1653">
<infon key="score">0.99879754</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:09:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="73202" length="7"/>
<text>calcium</text>
</annotation>
<annotation id="2058">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:19:10Z</infon>
<location offset="73214" length="14"/>
<text>coordinated by</text>
</annotation>
<annotation id="1654">
<infon key="score">0.99950826</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73229" length="5"/>
<text>Glu31</text>
</annotation>
<annotation id="1655">
<infon key="score">0.99950147</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:14:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73239" length="5"/>
<text>Glu34</text>
</annotation>
<annotation id="1656">
<infon key="score">0.99401796</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="73257" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1657">
<infon key="score">0.9978822</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:09:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73266" length="9"/>
<text>structure</text>
</annotation>
<annotation id="1658">
<infon key="score">0.99807453</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73288" length="10"/>
<text>entry site</text>
</annotation>
<annotation id="1659">
<infon key="score">0.99386215</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73330" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1660">
<infon key="score">0.999099</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="73376" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1661">
<infon key="score">0.99562365</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73444" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1662">
<infon key="score">0.9986205</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="73517" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1663">
<infon key="score">0.99952245</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73525" length="5"/>
<text>His57</text>
</annotation>
<annotation id="1664">
<infon key="score">0.9995121</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73532" length="5"/>
<text>Glu61</text>
</annotation>
<annotation id="1665">
<infon key="score">0.9995173</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73542" length="5"/>
<text>Glu64</text>
</annotation>
<annotation id="1666">
<infon key="score">0.9983008</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73607" length="22"/>
<text>mineralization surface</text>
</annotation>
<annotation id="1667">
<infon key="score">0.8620233</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="73641" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1668">
<infon key="score">0.99607795</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73651" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1669">
<infon key="score">0.8689013</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="73660" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1670">
<infon key="score">0.9678278</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73703" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1671">
<infon key="score">0.9975959</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73730" length="11"/>
<text>exit portal</text>
</annotation>
<annotation id="1672">
<infon key="score">0.9972073</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:17:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="73746" length="19"/>
<text>mineralization site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>73772</offset>
<text>The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B). Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins. While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell. Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme. TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).</text>
<annotation id="1673">
<infon key="score">0.9991721</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="73859" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1859">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:18:33Z</infon>
<location offset="73887" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1674">
<infon key="score">0.9978358</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="73937" length="4"/>
<text>iron</text>
</annotation>
<annotation id="2178">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<location offset="73962" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="1675">
<infon key="score">0.9787772</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:22:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="74053" length="28"/>
<text>biochemical characterization</text>
</annotation>
<annotation id="1676">
<infon key="score">0.9992699</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74085" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1677">
<infon key="score">0.99894625</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74177" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1678">
<infon key="score">0.9988219</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74214" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1908">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="74245" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1679">
<infon key="score">0.9991887</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="74255" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1680">
<infon key="score">0.9990702</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74272" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1681">
<infon key="score">0.9989749</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74294" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1682">
<infon key="score">0.9391922</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74332" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1683">
<infon key="score">0.5729172</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74343" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1684">
<infon key="score">0.98972017</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="74399" length="6"/>
<text>cavity</text>
</annotation>
<annotation id="1685">
<infon key="score">0.8813761</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74413" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1686">
<infon key="score">0.9983045</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="74424" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1687">
<infon key="score">0.9983613</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:47:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="74435" length="17"/>
<text>ferroxidase assay</text>
</annotation>
<annotation id="1688">
<infon key="score">0.9979518</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74477" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1689">
<infon key="score">0.9949738</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74488" length="16"/>
<text>nanocompartments</text>
</annotation>
<annotation id="1690">
<infon key="score">0.9991097</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74529" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1691">
<infon key="score">0.36210647</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:58:45Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74568" length="13"/>
<text>nanoparticles</text>
</annotation>
<annotation id="1692">
<infon key="score">0.971039</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74607" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1693">
<infon key="score">0.9990445</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74650" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1694">
<infon key="score">0.9991709</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74671" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1695">
<infon key="score">0.9986847</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:52:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="74686" length="3"/>
<text>TEM</text>
</annotation>
<annotation id="1696">
<infon key="score">0.99640995</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74760" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1697">
<infon key="score">0.99721414</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74791" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1698">
<infon key="score">0.98311573</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:58:50Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74802" length="15"/>
<text>nanocompartment</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig14.jpg</infon>
<infon key="id">fig14</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>74854</offset>
<text>Model of iron oxidation in encapsulin nanocompartments.</text>
<annotation id="1699">
<infon key="score">0.99905115</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="74863" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1700">
<infon key="score">0.9947168</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74881" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1701">
<infon key="score">0.99186254</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:47:29Z</infon>
<infon key="identifier">GO:</infon>
<location offset="74892" length="16"/>
<text>nanocompartments</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig14.jpg</infon>
<infon key="id">fig14</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>74910</offset>
<text>(A) Model of EncFtnsH docking to the encapsulin shell. A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface. The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT. Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively). The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron. The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment. As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.</text>
<annotation id="1702">
<infon key="score">0.9959959</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74923" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="2157">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:29Z</infon>
<location offset="74932" length="7"/>
<text>docking</text>
</annotation>
<annotation id="1703">
<infon key="score">0.47499946</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="74947" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1704">
<infon key="score">0.9856828</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="74958" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1705">
<infon key="score">0.99869543</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="74974" length="8"/>
<text>pentamer</text>
</annotation>
<annotation id="1706">
<infon key="score">0.99862695</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:36:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="74990" length="11"/>
<text>icosahedral</text>
</annotation>
<annotation id="1707">
<infon key="score">0.9982567</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="75002" length="11"/>
<text>T. maritima</text>
</annotation>
<annotation id="1708">
<infon key="score">0.7239029</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75014" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1709">
<infon key="score">0.99784875</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="75025" length="9"/>
<text>structure</text>
</annotation>
<annotation id="2196">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:59Z</infon>
<location offset="75085" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2197">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:11Z</infon>
<location offset="75096" length="21"/>
<text>localization sequence</text>
</annotation>
<annotation id="1710">
<infon key="score">0.99601483</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75121" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1711">
<infon key="score">0.99636465</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75185" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1712">
<infon key="score">0.9944976</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75192" length="8"/>
<text>subunits</text>
</annotation>
<annotation id="2181">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:44Z</infon>
<location offset="75235" length="22"/>
<text>localization sequences</text>
</annotation>
<annotation id="1713">
<infon key="score">0.99331784</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="75272" length="9"/>
<text>Alignment</text>
</annotation>
<annotation id="1714">
<infon key="score">0.9935847</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75285" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1715">
<infon key="score">0.9828522</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:14:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75318" length="15"/>
<text>central channel</text>
</annotation>
<annotation id="1716">
<infon key="score">0.9973459</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:14:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75353" length="4"/>
<text>pore</text>
</annotation>
<annotation id="1717">
<infon key="score">0.99818194</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="75370" length="8"/>
<text>pentamer</text>
</annotation>
<annotation id="1718">
<infon key="score">0.98649895</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75432" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1719">
<infon key="score">0.80627346</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75450" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2182">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:02:59Z</infon>
<location offset="75461" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1720">
<infon key="score">0.8879431</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75524" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2183">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:15Z</infon>
<location offset="75535" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1721">
<infon key="score">0.9398855</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="75595" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1722">
<infon key="score">0.48444474</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75632" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1723">
<infon key="score">0.9990338</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="75708" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1724">
<infon key="score">0.9990761</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="75739" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1725">
<infon key="score">0.7208568</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75765" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1726">
<infon key="score">0.94934714</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75776" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1727">
<infon key="score">0.53844655</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75786" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1728">
<infon key="score">0.8039653</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75793" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1729">
<infon key="score">0.9098615</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75856" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2187">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:16Z</infon>
<location offset="75867" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1730">
<infon key="score">0.947138</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:43Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75887" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1731">
<infon key="score">0.9990113</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="75918" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1732">
<infon key="score">0.9990713</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:50Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="75948" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1733">
<infon key="score">0.6161996</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="75975" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1734">
<infon key="score">0.86503077</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="75986" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1735">
<infon key="score">0.9974375</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76012" length="18"/>
<text>metal binding site</text>
</annotation>
<annotation id="1736">
<infon key="score">0.9860432</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76042" length="15"/>
<text>central channel</text>
</annotation>
<annotation id="1737">
<infon key="score">0.9866344</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76061" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1738">
<infon key="score">0.9989586</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:20:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76071" length="10"/>
<text>entry site</text>
</annotation>
<annotation id="1739">
<infon key="score">0.7974093</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76113" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1740">
<infon key="score">0.9989891</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:19:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="76132" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1741">
<infon key="score">0.88258153</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76225" length="10"/>
<text>encapsulin</text>
</annotation>
</passage>
<passage>
<infon key="file">elife-18972-fig14.jpg</infon>
<infon key="id">fig14</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>76242</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.034</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>76289</offset>
<text>Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT) shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A). Thus, it appears that the EncFtn decamer is the physiological state of this protein. This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn. A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron. The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment. This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.</text>
<annotation id="1742">
<infon key="score">0.99638414</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:22:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="76289" length="7"/>
<text>Docking</text>
</annotation>
<annotation id="1743">
<infon key="score">0.99840146</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76301" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1744">
<infon key="score">0.9968444</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:20:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76309" length="9"/>
<text>structure</text>
</annotation>
<annotation id="1745">
<infon key="score">0.9991373</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76322" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1746">
<infon key="score">0.9986046</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76340" length="8"/>
<text>pentamer</text>
</annotation>
<annotation id="1747">
<infon key="score">0.9983079</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="76356" length="11"/>
<text>T. maritima</text>
</annotation>
<annotation id="1748">
<infon key="score">0.99425864</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76368" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2158">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:20:39Z</infon>
<location offset="76379" length="10"/>
<text>Tmari_0786</text>
</annotation>
<annotation id="1749">
<infon key="score">0.9992826</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76437" length="21"/>
<text>C-terminal extensions</text>
</annotation>
<annotation id="1750">
<infon key="score">0.9988845</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76466" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1751">
<infon key="score">0.99812025</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:20:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76475" length="9"/>
<text>structure</text>
</annotation>
<annotation id="1752">
<infon key="score">0.87305796</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76509" length="22"/>
<text>localization sequences</text>
</annotation>
<annotation id="1753">
<infon key="score">0.99905574</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:20:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76537" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="1754">
<infon key="score">0.9951746</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76550" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1755">
<infon key="score">0.99610835</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76609" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1756">
<infon key="score">0.99860805</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="76616" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1757">
<infon key="score">0.99918133</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76699" length="12"/>
<text>central ring</text>
</annotation>
<annotation id="1758">
<infon key="score">0.9992908</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76715" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1759">
<infon key="score">0.998437</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76741" length="4"/>
<text>pore</text>
</annotation>
<annotation id="1760">
<infon key="score">0.9698333</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76784" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1761">
<infon key="score">0.99626005</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76795" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1762">
<infon key="score">0.9989838</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="76851" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1763">
<infon key="score">0.9633987</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76865" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1764">
<infon key="score">0.99898756</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="76892" length="11"/>
<text>active site</text>
</annotation>
<annotation id="1765">
<infon key="score">0.9991918</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76907" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1766">
<infon key="score">0.98662937</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="76935" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1767">
<infon key="score">0.9967763</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:58:59Z</infon>
<infon key="identifier">GO:</infon>
<location offset="76946" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1768">
<infon key="score">0.9652231</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="76970" length="8"/>
<text>ferritin</text>
</annotation>
<annotation id="1769">
<infon key="score">0.99120015</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="76979" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1770">
<infon key="score">0.85045284</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77076" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1771">
<infon key="score">0.999037</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="77115" length="4"/>
<text>iron</text>
</annotation>
<annotation id="2054">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:18:27Z</infon>
<location offset="77125" length="11"/>
<text>presence of</text>
</annotation>
<annotation id="1772">
<infon key="score">0.79746157</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:56:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77142" length="4"/>
<text>FOCs</text>
</annotation>
<annotation id="1773">
<infon key="score">0.99688536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T13:14:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77151" length="8"/>
<text>EncFtnsH</text>
</annotation>
<annotation id="1774">
<infon key="score">0.998533</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:53:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="77160" length="7"/>
<text>decamer</text>
</annotation>
<annotation id="1775">
<infon key="score">0.9973793</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:36:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="77190" length="11"/>
<text>icosahedral</text>
</annotation>
<annotation id="1776">
<infon key="score">0.9507636</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77202" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1777">
<infon key="score">0.99152315</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="77213" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1778">
<infon key="score">0.9986066</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="77247" length="9"/>
<text>decameric</text>
</annotation>
<annotation id="1779">
<infon key="score">0.9985454</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77257" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1780">
<infon key="score">0.9988722</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="77355" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1781">
<infon key="score">0.99581426</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:59:04Z</infon>
<infon key="identifier">GO:</infon>
<location offset="77393" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1909">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:44:52Z</infon>
<location offset="77503" length="9"/>
<text>classical</text>
</annotation>
<annotation id="1782">
<infon key="score">0.99890363</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:28:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="77513" length="9"/>
<text>ferritins</text>
</annotation>
<annotation id="1783">
<infon key="score">0.37426853</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77537" length="4"/>
<text>FOCs</text>
</annotation>
<annotation id="1784">
<infon key="score">0.9965545</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="77565" length="8"/>
<text>nanocage</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>77576</offset>
<text>Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C). The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1). The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs. We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).</text>
<annotation id="1785">
<infon key="score">0.9957445</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="77580" length="15"/>
<text>structural data</text>
</annotation>
<annotation id="1786">
<infon key="score">0.997255</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="77610" length="22"/>
<text>biochemical and ICP-MS</text>
</annotation>
<annotation id="1787">
<infon key="score">0.944305</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77683" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1788">
<infon key="score">0.9900916</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:59:19Z</infon>
<infon key="identifier">GO:</infon>
<location offset="77694" length="14"/>
<text>iron-megastore</text>
</annotation>
<annotation id="1789">
<infon key="score">0.998469</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:29:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="77727" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="1790">
<infon key="score">0.99831057</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:50:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="77752" length="11"/>
<text>T. maritima</text>
</annotation>
<annotation id="1791">
<infon key="score">0.6409168</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77764" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1792">
<infon key="score">0.8687022</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77775" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1793">
<infon key="score">0.9972558</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77795" length="23"/>
<text>negatively charged pore</text>
</annotation>
<annotation id="1794">
<infon key="score">0.9991125</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:56:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="77854" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1795">
<infon key="score">0.8829711</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77868" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1796">
<infon key="score">0.97599155</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77922" length="23"/>
<text>negatively charged hole</text>
</annotation>
<annotation id="1797">
<infon key="score">0.9972698</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="77953" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1798">
<infon key="score">0.9992531</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="77960" length="4"/>
<text>ring</text>
</annotation>
<annotation id="1799">
<infon key="score">0.99906075</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:15:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78006" length="19"/>
<text>metal-binding sites</text>
</annotation>
<annotation id="1800">
<infon key="score">0.99935883</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:04:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78049" length="4"/>
<text>ring</text>
</annotation>
<annotation id="2159">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:55Z</infon>
<location offset="78055" length="14"/>
<text>Glu31/34-sites</text>
</annotation>
<annotation id="1801">
<infon key="score">0.9990451</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:21:58Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78090" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1802">
<infon key="score">0.9981737</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:15:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78135" length="4"/>
<text>FOCs</text>
</annotation>
<annotation id="1803">
<infon key="score">0.9990736</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:22:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78174" length="4"/>
<text>Fe2+</text>
</annotation>
<annotation id="1804">
<infon key="score">0.9990438</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:22:13Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78182" length="4"/>
<text>Fe3+</text>
</annotation>
<annotation id="1805">
<infon key="score">0.9898706</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78205" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1806">
<infon key="score">0.9946569</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78260" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1807">
<infon key="score">0.9892139</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:15:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78274" length="14"/>
<text>substrate site</text>
</annotation>
<annotation id="1808">
<infon key="score">0.9989343</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78303" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1809">
<infon key="score">0.9987573</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:15:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78337" length="25"/>
<text>weakly coordinating sites</text>
</annotation>
<annotation id="1810">
<infon key="score">0.9995809</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="78406" length="5"/>
<text>His57</text>
</annotation>
<annotation id="1811">
<infon key="score">0.9995735</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="78413" length="5"/>
<text>Glu61</text>
</annotation>
<annotation id="1812">
<infon key="score">0.9995696</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T14:12:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="78423" length="5"/>
<text>Glu64</text>
</annotation>
<annotation id="1813">
<infon key="score">0.9990777</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:56:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78456" length="12"/>
<text>ferrihydrite</text>
</annotation>
<annotation id="1814">
<infon key="score">0.9501573</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="78532" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="2188">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T18:03:16Z</infon>
<location offset="78543" length="15"/>
<text>nanocompartment</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>78575</offset>
<text>Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store. Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.</text>
<annotation id="1815">
<infon key="score">0.99308926</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:22:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="78615" length="9"/>
<text>structure</text>
</annotation>
<annotation id="1816">
<infon key="score">0.9980296</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:22:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="78660" length="43"/>
<text>encapsulin-associated ferritin-like protein</text>
</annotation>
<annotation id="1817">
<infon key="score">0.9901558</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="78791" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1818">
<infon key="score">0.9713966</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:46:47Z</infon>
<infon key="identifier">GO:</infon>
<location offset="78802" length="8"/>
<text>nanocage</text>
</annotation>
<annotation id="1819">
<infon key="score">0.9989982</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78824" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1820">
<infon key="score">0.99803776</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T12:00:08Z</infon>
<infon key="identifier">GO:</infon>
<location offset="78856" length="10"/>
<text>EncFtn-Enc</text>
</annotation>
<annotation id="1821">
<infon key="score">0.9541096</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:59:11Z</infon>
<infon key="identifier">GO:</infon>
<location offset="78867" length="15"/>
<text>nanocompartment</text>
</annotation>
<annotation id="1822">
<infon key="score">0.99910176</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78939" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1823">
<infon key="score">0.97536135</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="78956" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1824">
<infon key="score">0.99871886</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:33:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="78967" length="5"/>
<text>shell</text>
</annotation>
<annotation id="1825">
<infon key="score">0.99908686</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="78991" length="4"/>
<text>iron</text>
</annotation>
<annotation id="1826">
<infon key="score">0.9758664</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:44Z</infon>
<infon key="identifier">PR:</infon>
<location offset="79013" length="6"/>
<text>EncFtn</text>
</annotation>
<annotation id="1827">
<infon key="score">0.9930814</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:54:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="79020" length="3"/>
<text>FOC</text>
</annotation>
<annotation id="1828">
<infon key="score">0.99498945</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:30:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="79071" length="10"/>
<text>encapsulin</text>
</annotation>
<annotation id="1829">
<infon key="score">0.9556411</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T17:59:24Z</infon>
<infon key="identifier">GO:</infon>
<location offset="79082" length="15"/>
<text>nanocompartment</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>79099</offset>
<text>Materials and methods</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>79121</offset>
<text>Cloning</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>79129</offset>
<text>Genes of interest were amplified by PCR using R. rubrum ATCC 11,170 genomic DNA (DSMZ) as the template and KOD Hot Start DNA Polymerase (Novagen). Primers used in this study are listed in Supplementary file 2. PCR products were visualized in 0.8% agarose gel stained with SYBR Safe (Life Technologies, UK). Fragments of interest were purified by gel extraction (Qiagen, UK) before digestion by endonuclease restriction enzymes (Thermo Fisher Scientific, UK) at 37°C for 1 hr, followed by ligation with similarly digested vector pET-28a(+) or pACYCDuet-1 at room temperature for 1 hr. Ligation product was transformed into chemically competent Escherichia coli Top10 cells and screened against 50 ng/μl kanamycin for pET-28a(+) based constructs or 34 ng/μl chloramphenicol for pACYCDuet-1 based constructs. DNA insertion was confirmed through Sanger sequencing (Edinburgh Genomics, The University of Edinburgh, UK). Sequence verified constructs were transformed into E. coli BL21(DE3) or Tuner(DE3) for protein production. Alternatively, plasmids transformed into E. coli B834(DE3) cells were cultured in selenomethionine medium.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>80264</offset>
<text>Protein production and purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>80300</offset>
<text>A single colony of E. coli BL21(DE3) or Tuner(DE3) cells, transformed with protein expression plasmid, was transferred into 10 ml LB medium, or M9 minimal medium (MM), supplemented with appropriate antibiotic, and incubated overnight at 37 °C with 200 rpm shaking. The overnight pre-culture was then inoculated into 1 liter of LB medium and incubated at 37 °C with 200 rpm shaking. Recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18°C for overnight incubation. Cells were pelleted by centrifugation at 4000 g for 20 min at 4 °C, and resuspended 10-fold (volume per gram of cell pellet) in PBS to wash cells before a second centrifugation step. Cells were resuspended in 10-times (v/w) of appropriate lysis buffer for the purification method used (see details of buffers below) and lysed by sonication on ice, with ten cycles of 30-second burst of sonication at 10 µm amplitude and 30 s of cooling. Cell lysate was clarified by centrifugation at 20,000 x g, 30 min, 4 °C; followed by filtration using a 0.22 µM syringe filter (Millipore, UK).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>81438</offset>
<text>Selenomethionine labelled protein was produced by growing a single colony of E. coli B834 (DE3) cells transformed with protein expression plasmids in 100 ml LB medium supplemented with appropriate antibiotic overnight at 37 °C with shaking at 200 rpm. The overnight pre-culture was pelleted by centrifugation 3,000 x g, 4 °C, 15 min and washed twice with M9 minimal medium. The washed cells were transferred to 1 liter of SeMet medium, which contains M9 minimal medium, 40 mg/L of each L-amino acid (without methionine), 40 mg/L selenomethionine, 2 mM MgSO4, 0.4% (w/v) glucose and 1 mM Fe(NH4)2(SO4)2. Cells were incubated at 37 °C with 200 rpm shaking and recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18 °C for overnight incubation. Cells were harvested and lysed as above.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>82309</offset>
<text>His-tagged protein purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>82341</offset>
<text>Clarified cell lysate was loaded onto a 5 ml HisTrap column (GE Healthcare, UK) pre-equilibrated with HisA buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 8.0). Unbound proteins were washed from the column with HisA buffer. His-tagged proteins were then eluted by a step gradient of 50% HisA buffer and 50% HisB buffer (50 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole, pH 8.0). Fractions containing the protein of interest, as determined by 15% (w/v) acrylamide SDS-PAGE, were pooled before loading onto a gel-filtration column (HiLoad 16/600 Superdex 200, GE Healthcare) equilibrated with GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Fractions were subjected to 15% SDS-PAGE and those containing the protein of interest were pooled for further analysis.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>83115</offset>
<text>Sucrose gradient ultracentrifugation purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>83165</offset>
<text>Co-expressed encapsulin and EncFtn (EncFtn-Enc) and encapsulin protein were both purified according to the protocol used by M. Sutter. Briefly, EncFtn-Enc or encapsulin was expressed based on pACYCDuet-1 vector. The E. coli cells were grown, induced, harvested and sonicated in a similar way as described above. GF buffer used in this purification contains 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. To remove RNA contamination, the lysate was supplemented with 50 μg/ml RNase A and rotated at 10 rpm and room temperature for 2 hrs, followed by centrifugation at 34,000 x g and 4 °C for 20 min and filtering through 0.22 µM syringe filter. Proteins were pelleted through 38% (w/v) sucrose cushion by ultracentrifugation at 100,000 x g and 4 °C for 21 hrs. 10% - 50% (w/v) sucrose gradient ultracentrifugation was applied to further separate the proteins at 100,000 x g and 4 °C for 17 hrs. Protein was dialyzed against GF buffer to remove sucrose before being used in chemical assays or TEM.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>84163</offset>
<text>Transmission electron microscopy</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>84196</offset>
<text>TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation.</text>
<annotation id="2163">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="84273" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="2164">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="84993" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>85392</offset>
<text>Protein crystallization and X-ray data collection</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>85442</offset>
<text>EncFtnsH was purified by anion exchange and Superdex 200 size- exclusion chromatography and concentrated to 10 mg/ml (based on extinction coefficient calculation). Crystallization drops were set up using the hanging drop vapor diffusion method at 292 K. Glass coverslips were set up with 1–2 μl protein mixed with 1 μl well solution (0.14 M calcium acetate and 15% (w/v) PEG 3350) and sealed over 1 ml of well solution. Crystals appeared after 5 days and were harvested from the well using a LithoLoop (Molecular Dimensions Limited, UK), transferred briefly to a cryoprotection solution containing well solution supplemented with 1 mM FeSO4 (in 0.1% (v/v) HCl), 20% (v/v) PEG 200, and subsequently flash cooled in liquid nitrogen. Crystals of the EncFtnsHsingle mutations were produced in the same manner as for the EncFtnsH wild-type protein.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>86295</offset>
<text>All crystallographic datasets were collected on the macromolecular crystallography beamlines at Diamond Light Source (Didcot, UK) at 100 K using Pilatus 6M detectors. Diffraction data were integrated and scaled using XDS and symmetry related reflections were merged with Aimless . Data collection statistics are shown in Table 4. The resolution cut-off used for structure determination and refinement was determined based on the CC1/2 criterion proposed by.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>86754</offset>
<text>The structure of EncFtnsH was determined by molecular replacement using PDB ID: 3K6C as the search model, modified to match the sequence of the target protein using Chainsaw. A single solution comprising three decamers in the asymmetric unit was found by molecular replacement using Phaser. The initial model was rebuilt using Phenix.autobuild followed by cycles of refinement with Phenix.refine, with manual rebuilding and model inspection in Coot . The final model was refined with isotropic B-factors, torsional NCS restraints, and with anomalous group refinement. The model was validated using MolProbity. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL. Multiple sequence alignment of EncFtn and ferritin family proteins was performed using Clustal Omega Sievers and Higgins, 2014 and displayed with Espript 3.0. Model refinement statistics are shown in Table 4. The final models and experimental data are deposited in the PDB and diffraction image files are available at the Edinburgh DataShare repository.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>87828</offset>
<text>Horse spleen apoferritin preparation</text>
<annotation id="2165">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="87841" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>87865</offset>
<text>Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5). Protein was dialyzed against 1 liter MOPS buffer in room temperature for two days before buffer exchanging to GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) in a vivaspin column with 5 kDa cut-off (Sartorius, UK) for several times. Fe content of apoferritin was detected using ferrozine assay. Protein concentration was determined using Pierce Microplate BCA Protein Assay Kit. Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay. Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer.</text>
<annotation id="2166">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="87878" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="2167">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="88281" length="11"/>
<text>apoferritin</text>
</annotation>
<annotation id="2168">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="88413" length="11"/>
<text>Apoferritin</text>
</annotation>
<annotation id="2169">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="88499" length="11"/>
<text>Apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>88612</offset>
<text>Ferroxidase assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>88630</offset>
<text>1 mM and 200 µM Fe(NH4)2(SO4)2 stock solutions were prepared in 0.1% (v/v) HCl anaerobically. Protein solutions with 20 µM FOC were diluted from ~10 mg/ml frozen stock in GF buffer (50 mM Tris-HCl, pH 8.0 and 150 mM NaCl) anaerobically. Ferroxidase activity was initiated by adding 450 μl protein to 50 μl of acidic Fe(NH4)2(SO4)2 at the final concentration of 100 µM and 20 µM in the air, respectively. The ferroxidase activity was measured by monitoring the Fe3+ formation which gives rise to the change of the absorbance at 315 nm. Absorbance at 315 nm was recorded every second over 1800 s using a quartz cuvette in a JASCO V-730 UV/VIS spectrophotometer (JASCO Inc., Easton, MD). In recombinantly coexpressed nanocompartments the ratio of EncFtn to Enc was assumed as 2 to 1, assuming each of the twelve pentameric vertices of the icosahedral encapsulin were occupied with decameric EncFtn. The data are presented as the mean of three technical replicates with error bars indicating one standard deviation from the mean. Proteins used here were from a single preparation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>89725</offset>
<text>Iron loading capacity of ferritins</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>89760</offset>
<text>In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions. The protein concentration was determined using PierceMicroplate BCA assay kit (Thermo Fisher Scientific). The protein standard curve was plotted according to the manufacturer. The Fe content in the samples was determined using modified ferrozine assay. Briefly speaking, 100 μl protein sample was mixed with 100 μl mixture of equal volume of 1.4 M HCl and 4.5% (w/v) KMnO4 and incubated at 60 °C for 2 hrs. 20 μl of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in H2O) was added to the cooled tubes. 30 min later, 200 μl of the solution was transferred into a well of 96-well plate and the absorbance at 562 nm was measured on the plate reader Spectramax M5 (Molecular Devices, UK). The standard curve was plotted using various concentrations of FeCl3 (in 10 mM HCl) diluted in the gel-filtration buffer. Three technical repeats were performed for both the ferrozine and microplate BCA assays. Samples analyzed by ICP-MS were prepared in the same way by mixing protein and ferrous ions and desalting. The proteins used in the Fe loading experiment came from a single preparation.</text>
<annotation id="2170">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="89897" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>91345</offset>
<text>Peroxidase assay</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>91362</offset>
<text>The peroxidase activity of EncFtnsH was determined by measuring the oxidation of ortho-phenylenediamine (OP) by H2O2 . EncFtnsH decameric and monomeric fractions purified from MM were both used in the assay. Ortho-phenylenediamine was prepared as a 92.5 mM stock solution in 50 mM Tris-HCl (pH 8.0). 80, 70, 60, 50, 40, 30, 20 and 10 mM of OP were prepared by diluting the stock solution in the 50 mM Tris-HCl (pH 8.0). 100 μl of each diluted OP was added to a 96-well plate in 3 repeats. 1 μl of 32 µM protein was supplemented into each well to a final concentration of 160 nM, followed by the addition of 2 μl of 30% H2O2. After 15 min shaking in the dark, the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The peroxidase activity was measured by monitoring the absorbance at 490 nm in the SpectraMax M5 Microplate Reader (Molecular Devices).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>92223</offset>
<text>ICP-MS analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>92239</offset>
<text>Protein samples were diluted 50-fold into a solution of 2.5% HNO3 (Suprapur, Merck, UK) containing 20 µg/L Pt as internal standard. Matrix-matched elemental standards (containing analyte metal concentrations 0 – 1000 µg/L) were prepared by serial dilution from individual metal standard stocks (VWR) with identical solution compositions, including the internal standard. All standards and samples were analyzed by ICP-MS using a Thermo x-series instrument (Thermo Fisher Scientific) operating in collision cell mode (using 3.0 ml min-1 flow of 8% H2 in He as the collision gas). Isotopes 44Ca, 56Fe, 66Zn, 78Se, and 195Pt were monitored using the peak-jump method (100 sweeps, 25–30 ms dwell time on 5 channels per isotope, separated by 0.02 atomic mass units) in triplicate. The protein samples used in ICP-MS came from a single protein preparation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>93098</offset>
<text>Mass spectrometry analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>93125</offset>
<text>For native MS analysis, all protein samples were buffer exchanged into 100 mM ammonium acetate (pH 8.0; adjusted with dropwise addition of 1% ammonia solution) using Micro Biospin Chromatography Columns (Bio-Rad, UK) prior to analysis and the resulting protein samples were analyzed at a final concentration of ~5 µM (oligomer concentration). In order to obtain Fe-bound EncFtn, 100 µM or 300 µM of freshly prepared FeCl2 was added to apo-EncFtnsH (monomer peak) immediately prior to buffer exchange into 100 mM ammonium acetate (pH 8.0). Samples were analyzed on a quadrupole ion-mobility time of flight instrument (Synapt G2, Waters Corp., Manchester, UK), equipped with a nanomate nanoelectrospray infusion robot (Advion Biosciences, Ithaca, NY). Instrument parameters were tuned to preserve non-covalent protein complexes. After optimization, typical parameters were: nanoelectrospray voltage 1.54 kV; sample cone 50 V; extractor cone 0 V; trap collision voltage 4 V; source temperature 80°C; and source backing pressure 5.5 mbar. For improved mass resolution the sample cone was raised to 155 V. Ion mobility mass spectrometry (IM-MS) was performed using the travelling-wave mobility cell in the Synapt G2, employing nitrogen as the drift gas. Typically, the IMS wave velocity was set to 300 m/s; wave height to 15 V; and the IMS pressure was 1.8 mbar. All native MS experiments were performed on samples from two independent protein preparations. For collision cross section determination, IM-MS data was calibrated using denatured equine myoglobin and data was analyzed using Driftscope v2.5 and MassLynx v4.1 (Waters Corp., UK). Theoretical collision cross sections (CCS) were calculated from pdb files using IMPACT software v. 0.9.1. In order to obtain information on the topology of the EncFtnsH assembly, gas-phase dissociation of the Fe-associated EncFtnsH complex was achieved by increasing the sample cone and/or trap collision voltage prior to MS analysis.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>95109</offset>
<text>SEC-MALLS</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>95119</offset>
<text>Size-exclusion chromatography (ÄKTA-Micro; GE Healthcare) coupled to UV, static light scattering and refractive index detection (Viscotec SEC-MALS 20 and Viscotec RI Detector:VE3580; Malvern Instruments, UK) were used to determine the molecular mass of fractions decamer and monomer of EncFtnsH in solution individually. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient ε0.1%= 1.462 mg−1 ml-1 cm−1. 100 μl of 1.43 mgml-1 fractions of EncFtnsH decamer and 4.03 mg ml -1 fractions of EncFtnsH monomer were run individually on a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl at 22°C with a flow rate of 0.5 ml/min. Light scattering, refractive index (RI) and A280nm were analyzed by a homo-polymer model (OmniSEC software, v 5.1; Malvern Instruments) using the following parameters for fractions of decamer and monomer: the extinction coefficient (dA/dc) at 280 nm was 1.46 AU mg ml−1 and specific refractive index increment (dn/dc) was 0.185 ml g−1. The proteins analyzed by SEC-MALLS came from single protein preparation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>96289</offset>
<text>Metal binding analysis by PAGE</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>96320</offset>
<text>Recombinant EncFtnsH fractions at 50 µM concentration were incubated with one molar equivalent of metal ions at room temperature for 2 hrs. Half of each sample was mixed with 5 x native loading buffer (65 mM Tris-HCl, pH 8.5, 20% glycerol and 0.01% bromophenol blue) and run on non-denaturing PAGE gels (10% acrylamide) and run in Tris/glycine buffer, 200 V, 4 °C for 50 min. The remaining samples were left for an additional three hours prior to SDS-PAGE (15% acrylamide) analysis. SDS-PAGE gels were run at room temperature at 200 V, room temperature for 50 min. Gels were stained with Coomassie Brilliant Blue R250 and scanned after de-staining in water. The proteins used in this experiment came from single protein preparation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>97057</offset>
<text>Analytical size-exclusion chromatography</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>97098</offset>
<text>For analysis of the multimeric state of EncFtn proteins by analytical size-exclusion gel-filtration chromatography (AGF) 25 μl of 90 µM protein was loaded into Superdex 200 PC 3.2/30 column (GE Healthcare) at 15 °C with GF buffer running at 0.05 ml/min and pressure limit 0.45 MPa. In order to use AGF to determine how metal ions influence the assembly of EncFtnsH, 90 µM EncFtnsH monomer fractions were mixed with equal molar concentrations of metal ion solutions including FeSO4 in 0.1% (v/v) HCl, Fe(NH4)2(SO4)2, FeCl3, CoCl2, calcium acetate (CaAc), ZnSO4 and MnCl2 at room temperature for 2 hrs prior to AGF analysis. Protein samples without metal titration were also analyzed as a control group. Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions. The AGF results have been repeated twice using two independent preparations of protein, of which only one representative trace is presented in the paper.</text>
<annotation id="2179">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:22:04Z</infon>
<location offset="97980" length="10"/>
<text>absence of</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>98177</offset>
<text>Accession codes and datasets</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>98206</offset>
<text>Coordinates and structure factors for the structures presented in this paper have been deposited in the PDB under the following accession codes: EncFtnsH, 5DA5; EncFtnsH-E32A, 5L89; EncFtnsH-E62A, 5L8B; EncFtnsH-H65A, 5L8G (DOIs for X-ray diffraction image data are shown in Table 4). All MS datasets presented in this paper can be found, in the raw format at http://dx.doi.org/10.7488/ds/1449.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">title_1</infon>
<offset>98601</offset>
<text>Funding Information</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98621</offset>
<text>This paper was supported by the following grants:</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98671</offset>
<text> to Didi He.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98684</offset>
<text> to Sam Hughes, Kirsten Altenbach, David J Clarke.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98735</offset>
<text> to Emma Tarrant, Kevin J Waldron.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98771</offset>
<text> to David J Clarke, Jon Marles-Wright.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">paragraph</infon>
<offset>98811</offset>
<text> to Jon Marles-Wright.</text>
</passage>
<passage>
<infon key="section_type">ACK_FUND</infon>
<infon key="type">title_1</infon>
<offset>98835</offset>
<text>Additional information</text>
</passage>
<passage>
<infon key="section_type">COMP_INT</infon>
<infon key="type">title_1</infon>
<offset>98858</offset>
<text>Competing interests</text>
</passage>
<passage>
<infon key="section_type">COMP_INT</infon>
<infon key="type">footnote</infon>
<offset>98878</offset>
<text>The authors declare that no competing interests exist.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">title_1</infon>
<offset>98933</offset>
<text>Author contributions</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>98954</offset>
<text>DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99073</offset>
<text>SH, Acquisition of data, Analysis and interpretation of data.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99135</offset>
<text>SV-H, Acquisition of data, Analysis and interpretation of data.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99199</offset>
<text>AG, Acquisition of data, Drafting or revising the article.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99258</offset>
<text>KA, Acquisition of data, Contributed unpublished essential data or reagents.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99335</offset>
<text>ET, Acquisition of data, Analysis and interpretation of data.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99397</offset>
<text>CLM, Acquisition of data, Contributed unpublished essential data or reagents.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99475</offset>
<text>KJW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99572</offset>
<text>DJC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">footnote</infon>
<offset>99692</offset>
<text>JM-W, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">title_1</infon>
<offset>99813</offset>
<text>Additional files</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">title_2</infon>
<offset>99830</offset>
<text>Major datasets</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">paragraph</infon>
<offset>99845</offset>
<text>The following datasets were generated:</text>
</passage>
<passage>
<infon key="section_type">REF</infon>
<infon key="type">title</infon>
<offset>99884</offset>
<text>References</text>
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<infon key="updated_at">2023-09-19T18:03:16Z</infon>
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<annotation id="2192">
<infon key="type">complex_assembly</infon>
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<infon key="annotator">cleaner0</infon>
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<offset>104178</offset>
<text>10.7554/eLife.18972.048</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">title</infon>
<offset>104202</offset>
<text>Decision letter</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>104218</offset>
<text>Losick</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>104225</offset>
<text>Richard</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>104233</offset>
<text>In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>104517</offset>
<text>[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>104711</offset>
<text>Thank you for submitting your work entitled "Structural characterisation of an encapsulated ferritin provides insight into iron storage in bacterial nanocompartments" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>105289</offset>
<text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>105888</offset>
<text>Reviewer #1: </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>105902</offset>
<text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.</text>
<annotation id="1885">
<infon key="type">chemical</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:35:28Z</infon>
<location offset="106158" length="6"/>
<text>oxygen</text>
</annotation>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>106212</offset>
<text>2) Results:</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>106224</offset>
<text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>106364</offset>
<text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>106635</offset>
<text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system. Thus the significance of the experimental results in the paper are unclear.</text>
<annotation id="2171">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="106744" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>107164</offset>
<text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)</text>
<annotation id="2172">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T16:21:26Z</infon>
<location offset="107321" length="11"/>
<text>apoferritin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>107409</offset>
<text>Reviewer #2: </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>107423</offset>
<text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below:</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>108298</offset>
<text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed).</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>108754</offset>
<text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role?</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109070</offset>
<text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved?</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109185</offset>
<text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system.</text>
<annotation id="2155">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T15:12:22Z</infon>
<location offset="109219" length="11"/>
<text>mutagenesis</text>
</annotation>
<annotation id="1879">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T11:32:31Z</infon>
<location offset="109292" length="11"/>
<text>ferroxidase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109403</offset>
<text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109612</offset>
<text>10.7554/eLife.18972.049</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">title</infon>
<offset>109636</offset>
<text>Author response</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109652</offset>
<text>[Editors’ note: the author responses to the first round of peer review follow.]</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>109734</offset>
<text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>110334</offset>
<text>Reviewer #1: </text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>110348</offset>
<text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>110659</offset>
<text>The reviewer makes an excellent point here. To ascertain whether the iron in the assays forms ‘balls of rust’ we performed transmission electron microscopy on the ferroxidase reaction mixtures after completion of the reaction to assess the formation of free, or encapsulated iron minerals. We provide an additional supplemental figure (Figure 8—figure supplement 1) and discuss the observation of iron mineral crystals and nanoparticles in the main text, subsection “Ferroxidase activity”, last paragraph. We also attempted to use a commercial luminescence-based ROS detection kit on the reactions to address the possibility that H2O2 is produced as a reaction intermediate by the EncFtn protein. We found that the results from this particular kit were inconsistent between repeats, but for the benefit of the reviewer we provide a graph of the results obtained (see Author response image 1). These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.</text>
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<infon key="type">fig_caption</infon>
<offset>111792</offset>
<text>DOI:
http://dx.doi.org/10.7554/eLife.18972.037</text>
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<infon key="type">paragraph</infon>
<offset>111839</offset>
<text>We acknowledge that the reaction mechanism of the EncFtn merits further investigation in a follow up study.</text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>111947</offset>
<text>2) Results:</text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>111959</offset>
<text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>112100</offset>
<text>We acknowledge that the data for iron loading merits inclusion in the main text, we have now moved this data and other supplementary data tables to the main text.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>112263</offset>
<text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>112535</offset>
<text>One of the central arguments of our paper is the fact that the EncFtnsH monomer must dimerize to produce a functional ferroxidase active site and that its iron binding properties are highly divergent from those of the classical ferritin nanocages. We have added additional text to the manuscript to highlight these differences (Introduction, last paragraph, and Mass spectrometry section) and discuss the functional consequences at length.</text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>112975</offset>
<text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system! Thus the significance of the experimental results in the paper are unclear. </text>
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<offset>113505</offset>
<text>We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph). The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.</text>
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<text>nanocompartment</text>
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<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>113906</offset>
<text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.) </text>
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<infon key="type">paragraph</infon>
<offset>114152</offset>
<text>The data for the starting material are shown in Table 5. Control data for apoferritin have been added to this table and are illustrated in Figure 8. We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.</text>
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<text>nanocompartment</text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>114661</offset>
<text>Reviewer #2: </text>
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<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>114675</offset>
<text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below: </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>115551</offset>
<text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed). </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>116008</offset>
<text>The reviewer makes an interesting point about growth conditions and we acknowledge that production of the protein in LB medium leads to varying protein yields and monomer/decamer proportions. We therefore adopted the use of M9 minimal medium throughout the study to give better reproducibility, which also enables better control of metal ion availability than the complex LB medium. Given the fact that the protein is produced recombinantly in E. coli it is not particularly instructive to prove the in vivoproduction of the EncFtn multimer in this host. We have added a panel to Figure 3 to show the effect of protein concentration on multimerization in vitro(Figure 3C). Our mass spectrometry results show that the protein spontaneously multimerized in the presence of iron in vitroto form decameric species and that this is metal ion concentration dependent (Figure 7).</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>116881</offset>
<text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role? </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>117198</offset>
<text>We have put a comment in the Introduction to introduce R. rubrum (last paragraph). A preliminary study in the laboratory identified encapsulins in a preparation of lipid vesicles from R. rubrum containing chromatophores. We chose to follow up on these structures in this study. We do not feel this particular information is key to the central argument of the paper.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>117564</offset>
<text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved? </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>117680</offset>
<text>We have noted this in the Introduction of the manuscript.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>117738</offset>
<text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>117957</offset>
<text>To address this question we have produced three FOC mutants of the EncFtn protein and characterized these in solution, by mass spectrometry, and crystallographically (section: Mutagenesis of the EncFtnsH Ferroxidase center). We thank the reviewer for this suggestion as it highlighted the importance of the FOC residues for assembly and activity, and our new data has provided interesting insights into the EncFtn protein.</text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>118380</offset>
<text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another. </text>
</passage>
<passage>
<infon key="section_type">REVIEW_INFO</infon>
<infon key="type">paragraph</infon>
<offset>118590</offset>
<text>We now include a phylogenetic tree (Figure 13) and consider the question of ferritin evolution in the Discussion (first paragraph). None of the authors of this study are evolutionary biologists but we appreciate the difficulty inherent in tracing the history of protein folds, especially in bacterial lineages. We refer to previous studies in this section and make a suggestion that can be followed up in subsequent studies.</text>
</passage>
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<infon key="article-id_doi">10.1016/j.str.2016.06.020</infon>
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<infon key="article-id_publisher-id">S0969-2126(16)30167-8</infon>
<infon key="fpage">1599</infon>
<infon key="issue">9</infon>
<infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon>
<infon key="lpage">1605</infon>
<infon key="name_0">surname:Zebisch;given-names:Matthias</infon>
<infon key="name_1">surname:Jackson;given-names:Verity A.</infon>
<infon key="name_2">surname:Zhao;given-names:Yuguang</infon>
<infon key="name_3">surname:Jones;given-names:E. Yvonne</infon>
<infon key="notes">Published: August 11, 2016</infon>
<infon key="section_type">TITLE</infon>
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<infon key="volume">24</infon>
<infon key="year">2016</infon>
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<text>Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf</text>
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<text>Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.</text>
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<text>Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3812" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="97">
<infon key="score">0.9985933</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3821" length="3"/>
<text>Dkk</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3834</offset>
<text>Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.</text>
<annotation id="475">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:42:04Z</infon>
<location offset="3848" length="4"/>
<text>Krm1</text>
</annotation>
<annotation id="541">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:42:15Z</infon>
<location offset="3857" length="1"/>
<text>2</text>
</annotation>
<annotation id="98">
<infon key="score">0.9981333</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:53:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3863" length="29"/>
<text>type I transmembrane proteins</text>
</annotation>
<annotation id="99">
<infon key="score">0.99945766</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3907" length="10"/>
<text>ectodomain</text>
</annotation>
<annotation id="100">
<infon key="score">0.9392079</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3924" length="8"/>
<text>flexible</text>
</annotation>
<annotation id="101">
<infon key="score">0.9981252</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:42:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3933" length="16"/>
<text>cytoplasmic tail</text>
</annotation>
<annotation id="102">
<infon key="score">0.8183996</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:42:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3964" length="2"/>
<text>60</text>
</annotation>
<annotation id="103">
<infon key="score">0.6206278</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:42:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3967" length="2"/>
<text>75</text>
</annotation>
<annotation id="104">
<infon key="score">0.99932384</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3984" length="10"/>
<text>ectodomain</text>
</annotation>
<annotation id="479">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:07:04Z</infon>
<location offset="4086" length="7"/>
<text>Kringle</text>
</annotation>
<annotation id="105">
<infon key="score">0.9994307</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4102" length="2"/>
<text>KR</text>
</annotation>
<annotation id="106">
<infon key="score">0.9988023</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:07:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4123" length="3"/>
<text>WSC</text>
</annotation>
<annotation id="533">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:34Z</infon>
<location offset="4184" length="3"/>
<text>CUB</text>
</annotation>
<annotation id="107">
<infon key="score">0.6716569</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:08:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4199" length="24"/>
<text>approximately 70-residue</text>
</annotation>
<annotation id="108">
<infon key="score">0.98141634</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4224" length="6"/>
<text>linker</text>
</annotation>
<annotation id="109">
<infon key="score">0.99934775</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4244" length="3"/>
<text>CUB</text>
</annotation>
<annotation id="110">
<infon key="score">0.9992559</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4262" length="18"/>
<text>transmembrane span</text>
</annotation>
<annotation id="111">
<infon key="score">0.9983392</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4285" length="6"/>
<text>intact</text>
</annotation>
<annotation id="542">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:43:37Z</infon>
<location offset="4292" length="10"/>
<text>KR-WSC-CUB</text>
</annotation>
<annotation id="517">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="4358" length="3"/>
<text>Wnt</text>
</annotation>
<annotation id="112">
<infon key="score">0.999244</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4378" length="18"/>
<text>transmembrane span</text>
</annotation>
<annotation id="113">
<infon key="score">0.9991549</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:43:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4401" length="16"/>
<text>cytoplasmic tail</text>
</annotation>
<annotation id="543">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:08Z</infon>
<location offset="4441" length="3"/>
<text>GPI</text>
</annotation>
<annotation id="544">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:20Z</infon>
<location offset="4445" length="6"/>
<text>linker</text>
</annotation>
<annotation id="518">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="4470" length="3"/>
<text>Wnt</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>4490</offset>
<text>We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.</text>
<annotation id="114">
<infon key="score">0.9981673</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:44:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4595" length="10"/>
<text>structures</text>
</annotation>
<annotation id="115">
<infon key="score">0.9994917</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:07:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4652" length="3"/>
<text>WSC</text>
</annotation>
<annotation id="116">
<infon key="score">0.99919</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:00Z</infon>
<infon key="identifier">GO:</infon>
<location offset="4770" length="27"/>
<text>LRP6PE3PE4-DKK1CRD2-KRM1ECD</text>
</annotation>
<annotation id="117">
<infon key="score">0.5890333</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4840" length="3"/>
<text>Wnt</text>
</annotation>
<annotation id="118">
<infon key="score">0.6935159</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:08:21Z</infon>
<infon key="identifier">GO:</infon>
<location offset="4844" length="18"/>
<text>inhibitory complex</text>
</annotation>
<annotation id="119">
<infon key="score">0.99836403</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4877" length="4"/>
<text>Lrp6</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>4907</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>4915</offset>
<text>The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.</text>
<annotation id="120">
<infon key="score">0.99909973</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4949" length="20"/>
<text>extracellular domain</text>
</annotation>
<annotation id="121">
<infon key="score">0.97918665</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:53:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4973" length="3"/>
<text>Krm</text>
</annotation>
<annotation id="545">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:21Z</infon>
<location offset="4981" length="18"/>
<text>structural studies</text>
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<annotation id="546">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:46:05Z</infon>
<location offset="5076" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="547">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:46:16Z</infon>
<location offset="5080" length="3"/>
<text>ECD</text>
</annotation>
<annotation id="122">
<infon key="score">0.72899675</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5084" length="14"/>
<text>complexes with</text>
</annotation>
<annotation id="123">
<infon key="score">0.85313314</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:46:23Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5099" length="6"/>
<text>DKK1fl</text>
</annotation>
<annotation id="548">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:46:42Z</infon>
<location offset="5107" length="4"/>
<text>DKK1</text>
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<annotation id="549">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:00Z</infon>
<location offset="5111" length="11"/>
<text>Linker-CRD2</text>
</annotation>
<annotation id="550">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:15Z</infon>
<location offset="5128" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="551">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:24Z</infon>
<location offset="5132" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="124">
<infon key="score">0.96971107</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5174" length="14"/>
<text>gel filtration</text>
</annotation>
<annotation id="125">
<infon key="score">0.99780023</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5262" length="13"/>
<text>crystal forms</text>
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<annotation id="126">
<infon key="score">0.9982065</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5321" length="8"/>
<text>crystals</text>
</annotation>
<annotation id="127">
<infon key="score">0.99675816</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5352" length="4"/>
<text>KRM1</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>5366</offset>
<text>We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).</text>
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<infon key="score">0.99417144</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5369" length="6"/>
<text>solved</text>
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<infon key="score">0.9750115</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:47:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5380" length="9"/>
<text>structure</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:04Z</infon>
<location offset="5393" length="4"/>
<text>KRM1</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:15Z</infon>
<location offset="5397" length="3"/>
<text>ECD</text>
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<infon key="score">0.99771154</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5489" length="9"/>
<text>structure</text>
</annotation>
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<infon key="score">0.8854561</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5509" length="11"/>
<text>full-length</text>
</annotation>
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<infon key="score">0.6456017</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5543" length="5"/>
<text>small</text>
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<infon key="score">0.7668342</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5550" length="8"/>
<text>flexible</text>
</annotation>
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<location offset="5915" length="3"/>
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<location offset="5919" length="3"/>
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<location offset="5924" length="3"/>
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<location offset="5928" length="4"/>
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<location offset="6037" length="7"/>
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<infon key="updated_at">2023-09-19T08:51:04Z</infon>
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<location offset="6101" length="26"/>
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<location offset="6129" length="4"/>
<text>RMSD</text>
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<infon key="updated_at">2023-09-19T08:51:18Z</infon>
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<location offset="6184" length="9"/>
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<infon key="updated_at">2023-09-18T15:07:09Z</infon>
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<location offset="6218" length="3"/>
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<infon key="updated_at">2023-09-19T09:56:55Z</infon>
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<location offset="6253" length="9"/>
<text>structure</text>
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<infon key="updated_at">2023-09-19T08:51:25Z</infon>
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<location offset="6286" length="8"/>
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<infon key="updated_at">2023-09-19T08:51:22Z</infon>
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<location offset="6300" length="35"/>
<text>β1-β5-β3-β4-β2 antiparallel β sheet</text>
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<infon key="updated_at">2023-09-19T08:51:30Z</infon>
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<location offset="6349" length="7"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:56:58Z</infon>
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<location offset="6362" length="9"/>
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<infon key="updated_at">2023-09-19T08:51:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6388" length="5"/>
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<infon key="updated_at">2023-09-19T10:08:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6420" length="17"/>
<text>disulfide bridges</text>
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<infon key="updated_at">2023-09-19T10:14:03Z</infon>
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<location offset="6439" length="4"/>
<text>C122</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:14:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6444" length="4"/>
<text>C186</text>
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<infon key="updated_at">2023-09-19T10:14:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6450" length="4"/>
<text>C147</text>
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<location offset="6455" length="4"/>
<text>C167</text>
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<infon key="updated_at">2023-09-19T10:14:12Z</infon>
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<location offset="6461" length="4"/>
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<infon key="updated_at">2023-09-19T10:14:14Z</infon>
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<location offset="6466" length="4"/>
<text>C169</text>
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<location offset="6472" length="4"/>
<text>C190</text>
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<infon key="updated_at">2023-09-19T10:14:19Z</infon>
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<location offset="6477" length="4"/>
<text>C198</text>
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<location offset="6494" length="15"/>
<text>PDBeFold server</text>
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<infon key="updated_at">2023-09-19T09:58:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6564" length="18"/>
<text>PAN module domains</text>
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<infon key="updated_at">2023-09-19T09:56:16Z</infon>
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<location offset="6619" length="24"/>
<text>hepatocyte growth factor</text>
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<infon key="updated_at">2023-09-19T09:56:24Z</infon>
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<location offset="6645" length="3"/>
<text>HGF</text>
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<infon key="updated_at">2023-09-19T10:03:16Z</infon>
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<location offset="6668" length="10"/>
<text>superposes</text>
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<infon key="updated_at">2023-09-19T08:51:09Z</infon>
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<location offset="6687" length="4"/>
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<location offset="6736" length="3"/>
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<infon key="updated_at">2023-09-19T08:51:54Z</infon>
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<location offset="6757" length="19"/>
<text>glycosylation sites</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:52:12Z</infon>
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<location offset="6815" length="16"/>
<text>electron density</text>
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<infon key="updated_at">2023-09-19T08:52:20Z</infon>
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<location offset="6839" length="4"/>
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<infon key="identifier">DUMMY:</infon>
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<infon key="identifier">CHEBI:</infon>
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<text>coordinated by</text>
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<infon key="updated_at">2023-09-19T08:52:27Z</infon>
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<location offset="7054" length="4"/>
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<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-19T10:04:02Z</infon>
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<location offset="7085" length="19"/>
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<infon key="identifier">DUMMY:</infon>
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<location offset="7288" length="13"/>
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<infon key="updated_at">2023-09-19T10:06:03Z</infon>
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<infon key="updated_at">2023-09-19T09:58:11Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:05:38Z</infon>
<location offset="7380" length="13"/>
<text>crystal forms</text>
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<annotation id="558">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:11Z</infon>
<location offset="7434" length="5"/>
<text>CUB_C</text>
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<annotation id="190">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:15Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7450" length="5"/>
<text>Tsg-6</text>
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<annotation id="191">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7475" length="10"/>
<text>superposes</text>
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<annotation id="559">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:33Z</infon>
<location offset="7491" length="3"/>
<text>KRM</text>
</annotation>
<annotation id="560">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:41Z</infon>
<location offset="7494" length="3"/>
<text>CUB</text>
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<annotation id="192">
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<infon key="updated_at">2023-09-19T08:51:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7506" length="4"/>
<text>RMSD</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>7578</offset>
<text>A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains. Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain. The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A). Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.</text>
<annotation id="193">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:58Z</infon>
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<location offset="7580" length="13"/>
<text>superposition</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7607" length="4"/>
<text>KRM1</text>
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<annotation id="195">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:03Z</infon>
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<location offset="7612" length="10"/>
<text>structures</text>
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<infon key="updated_at">2023-09-19T10:04:45Z</infon>
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<location offset="7803" length="17"/>
<text>Ca2+ binding site</text>
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<annotation id="197">
<infon key="score">0.98170817</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:20Z</infon>
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<location offset="7824" length="24"/>
<text>crystal forms II and III</text>
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<annotation id="198">
<infon key="score">0.9801446</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:58:17Z</infon>
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<location offset="7853" length="4"/>
<text>loop</text>
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<annotation id="199">
<infon key="score">0.9993906</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7877" length="2"/>
<text>KR</text>
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<annotation id="200">
<infon key="score">0.9988738</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7892" length="5"/>
<text>F207S</text>
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<annotation id="201">
<infon key="score">0.53296876</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7988" length="16"/>
<text>hydrophobic core</text>
</annotation>
<annotation id="202">
<infon key="score">0.998882</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8027" length="9"/>
<text>interface</text>
</annotation>
<annotation id="662">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:37Z</infon>
<location offset="8093" length="8"/>
<text>bound to</text>
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<annotation id="203">
<infon key="score">0.9980704</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8148" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="519">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="8184" length="3"/>
<text>Wnt</text>
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<annotation id="520">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="8202" length="3"/>
<text>Wnt</text>
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<annotation id="521">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="8223" length="3"/>
<text>Wnt</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>8251</offset>
<text>Low-Resolution Insight into Ternary Complex Formation</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>8305</offset>
<text>Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.</text>
<annotation id="204">
<infon key="score">0.9989608</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8305" length="18"/>
<text>Co-crystallization</text>
</annotation>
<annotation id="561">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:55:01Z</infon>
<location offset="8327" length="4"/>
<text>LRP6</text>
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<annotation id="562">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:55:12Z</infon>
<location offset="8331" length="6"/>
<text>PE3PE4</text>
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<annotation id="563">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:55:27Z</infon>
<location offset="8343" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="564">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:55:37Z</infon>
<location offset="8347" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="565">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:55:52Z</infon>
<location offset="8357" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="566">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:03Z</infon>
<location offset="8361" length="3"/>
<text>PE1</text>
</annotation>
<annotation id="205">
<infon key="score">0.9981754</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8395" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="522">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="8453" length="3"/>
<text>Wnt</text>
</annotation>
<annotation id="206">
<infon key="score">0.5106511</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8471" length="3"/>
<text>Dkk</text>
</annotation>
<annotation id="207">
<infon key="score">0.9107751</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8507" length="4"/>
<text>flat</text>
</annotation>
<annotation id="567">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:40Z</infon>
<location offset="8512" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="568">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:50Z</infon>
<location offset="8516" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="663">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:00:11Z</infon>
<location offset="8530" length="8"/>
<text>binds to</text>
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<annotation id="208">
<infon key="score">0.99904746</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8543" length="17"/>
<text>third β propeller</text>
</annotation>
<annotation id="209">
<infon key="score">0.99905545</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="8564" length="4"/>
<text>LRP6</text>
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<annotation id="210">
<infon key="score">0.99858415</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8570" length="19"/>
<text>Mutational analyses</text>
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<annotation id="571">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:57:26Z</infon>
<location offset="8615" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="572">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:57:36Z</infon>
<location offset="8619" length="3"/>
<text>PE3</text>
</annotation>
<annotation id="569">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:57:04Z</infon>
<location offset="8639" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="570">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:57:14Z</infon>
<location offset="8643" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="211">
<infon key="score">0.9990976</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:59:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="8658" length="16"/>
<text>Krm binding site</text>
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<annotation id="212">
<infon key="score">0.9959565</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8697" length="3"/>
<text>Dkk</text>
</annotation>
<annotation id="573">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:59:53Z</infon>
<location offset="8718" length="9"/>
<text>receptors</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>8755</offset>
<text>To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).</text>
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<infon key="score">0.9977992</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:00:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8814" length="6"/>
<text>Lrp5/6</text>
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<annotation id="214">
<infon key="score">0.9974843</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8822" length="3"/>
<text>Dkk</text>
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<infon key="score">0.99199754</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8831" length="3"/>
<text>Krm</text>
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<annotation id="216">
<infon key="score">0.99846774</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:00:46Z</infon>
<infon key="identifier">GO:</infon>
<location offset="8852" length="25"/>
<text>LRP6PE3PE4-DKK1fl-KRM1ECD</text>
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<infon key="score">0.99831676</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="8889" length="22"/>
<text>crystallization trials</text>
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<infon key="score">0.9906544</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8913" length="16"/>
<text>Diffraction data</text>
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<infon key="score">0.9968412</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="8959" length="8"/>
<text>crystals</text>
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<infon key="score">0.6915508</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:19Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>diffraction</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:39Z</infon>
<location offset="9225" length="4"/>
<text>DKK1</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:01:49Z</infon>
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<text>CRD2</text>
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<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:00:12Z</infon>
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<text>binds to</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9263" length="4"/>
<text>LRP6</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:50Z</infon>
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<text>PE3</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:24Z</infon>
<location offset="9272" length="11"/>
<text>β propeller</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:02:28Z</infon>
<location offset="9329" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="579">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:02:38Z</infon>
<location offset="9333" length="3"/>
<text>ECD</text>
</annotation>
<annotation id="666">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:00:33Z</infon>
<location offset="9349" length="7"/>
<text>bind on</text>
</annotation>
<annotation id="576">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:02:01Z</infon>
<location offset="9378" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="577">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:02:13Z</infon>
<location offset="9382" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="222">
<infon key="score">0.9995098</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9401" length="2"/>
<text>KR</text>
</annotation>
<annotation id="223">
<infon key="score">0.9993789</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:07:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9408" length="3"/>
<text>WSC</text>
</annotation>
<annotation id="224">
<infon key="score">0.9972345</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9497" length="15"/>
<text>crystallization</text>
</annotation>
<annotation id="225">
<infon key="score">0.9782202</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9528" length="7"/>
<text>density</text>
</annotation>
<annotation id="226">
<infon key="score">0.9987436</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:58:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9561" length="4"/>
<text>CRD1</text>
</annotation>
<annotation id="227">
<infon key="score">0.7814616</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:03:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9573" length="13"/>
<text>domain linker</text>
</annotation>
<annotation id="228">
<infon key="score">0.9993906</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:03:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9588" length="1"/>
<text>L</text>
</annotation>
<annotation id="229">
<infon key="score">0.99893695</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9612" length="4"/>
<text>CRD2</text>
</annotation>
<annotation id="230">
<infon key="score">0.9989029</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9620" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="231">
<infon key="score">0.9977888</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9667" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="232">
<infon key="score">0.9989211</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9676" length="25"/>
<text>surface plasmon resonance</text>
</annotation>
<annotation id="233">
<infon key="score">0.9985241</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9703" length="3"/>
<text>SPR</text>
</annotation>
<annotation id="234">
<infon key="score">0.9858543</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9736" length="8"/>
<text>affinity</text>
</annotation>
<annotation id="235">
<infon key="score">0.99905944</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9753" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="236">
<infon key="score">0.99898297</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="9765" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="581">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:03:59Z</infon>
<location offset="9786" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="582">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:09Z</infon>
<location offset="9790" length="3"/>
<text>ECD</text>
</annotation>
<annotation id="237">
<infon key="score">0.9979876</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9809" length="11"/>
<text>SUMO fusion</text>
</annotation>
<annotation id="580">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:03:44Z</infon>
<location offset="9824" length="10"/>
<text>DKK1L-CRD2</text>
</annotation>
<annotation id="238">
<infon key="score">0.98279107</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9873" length="8"/>
<text>affinity</text>
</annotation>
<annotation id="239">
<infon key="score">0.9980215</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9898" length="11"/>
<text>SUMO fusion</text>
</annotation>
<annotation id="240">
<infon key="score">0.9986412</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="9913" length="10"/>
<text>DKK1CRD1-L</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10014</offset>
<text>Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).</text>
<annotation id="241">
<infon key="score">0.9988619</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10027" length="19"/>
<text>DKK1-KRM1 interface</text>
</annotation>
<annotation id="242">
<infon key="score">0.9965975</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10085" length="18"/>
<text>polar interactions</text>
</annotation>
<annotation id="243">
<infon key="score">0.99685144</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10117" length="20"/>
<text>hydrophobic contacts</text>
</annotation>
<annotation id="244">
<infon key="score">0.99868906</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:08:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10155" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="245">
<infon key="score">0.9990559</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10198" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="246">
<infon key="score">0.9994848</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10252" length="4"/>
<text>R191</text>
</annotation>
<annotation id="247">
<infon key="score">0.9990158</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10260" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="248">
<infon key="score">0.99034667</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10280" length="11"/>
<text>salt bridge</text>
</annotation>
<annotation id="249">
<infon key="score">0.9994804</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10295" length="4"/>
<text>D125</text>
</annotation>
<annotation id="250">
<infon key="score">0.9994967</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10304" length="4"/>
<text>E162</text>
</annotation>
<annotation id="251">
<infon key="score">0.5185777</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10312" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="584">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:11:06Z</infon>
<location offset="10332" length="15"/>
<text>charge reversal</text>
</annotation>
<annotation id="252">
<infon key="score">0.9976808</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10358" length="5"/>
<text>mouse</text>
</annotation>
<annotation id="253">
<infon key="score">0.9989874</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:56:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10364" length="4"/>
<text>Dkk1</text>
</annotation>
<annotation id="254">
<infon key="score">0.9891629</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10370" length="5"/>
<text>mDkk1</text>
</annotation>
<annotation id="255">
<infon key="score">0.99904364</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10377" length="5"/>
<text>R197E</text>
</annotation>
<annotation id="256">
<infon key="score">0.9994855</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10442" length="4"/>
<text>K226</text>
</annotation>
<annotation id="257">
<infon key="score">0.998976</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10461" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="258">
<infon key="score">0.7484227</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10491" length="18"/>
<text>hydrophobic pocket</text>
</annotation>
<annotation id="259">
<infon key="score">0.88917637</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10528" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="260">
<infon key="score">0.9995097</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10543" length="4"/>
<text>Y108</text>
</annotation>
<annotation id="261">
<infon key="score">0.99953055</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10549" length="3"/>
<text>W94</text>
</annotation>
<annotation id="262">
<infon key="score">0.99950445</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10558" length="4"/>
<text>W106</text>
</annotation>
<annotation id="263">
<infon key="score">0.9966936</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10570" length="12"/>
<text>salt bridges</text>
</annotation>
<annotation id="264">
<infon key="score">0.9668133</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10607" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="265">
<infon key="score">0.9995011</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:09:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10612" length="3"/>
<text>D88</text>
</annotation>
<annotation id="266">
<infon key="score">0.99948114</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:09:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10620" length="3"/>
<text>D90</text>
</annotation>
<annotation id="585">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:11:12Z</infon>
<location offset="10634" length="15"/>
<text>charge reversal</text>
</annotation>
<annotation id="267">
<infon key="score">0.96642536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:14Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10670" length="5"/>
<text>mDkk1</text>
</annotation>
<annotation id="268">
<infon key="score">0.9990295</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10676" length="5"/>
<text>K232E</text>
</annotation>
<annotation id="269">
<infon key="score">0.9990246</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10736" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="270">
<infon key="score">0.99949574</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:09:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10741" length="4"/>
<text>S192</text>
</annotation>
<annotation id="271">
<infon key="score">0.7990252</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10783" length="3"/>
<text>Krm</text>
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<annotation id="272">
<infon key="score">0.99949014</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:09:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10848" length="4"/>
<text>D201</text>
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<annotation id="273">
<infon key="score">0.92195415</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10856" length="4"/>
<text>KRM1</text>
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<annotation id="274">
<infon key="score">0.9971021</infon>
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<infon key="updated_at">2023-09-19T09:06:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10871" length="14"/>
<text>hydrogen bonds</text>
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<annotation id="275">
<infon key="score">0.9994716</infon>
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<infon key="updated_at">2023-09-19T09:09:11Z</infon>
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<location offset="10939" length="4"/>
<text>S192</text>
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<infon key="updated_at">2023-09-19T09:08:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10945" length="5"/>
<text>mouse</text>
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<annotation id="277">
<infon key="score">0.99947363</infon>
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<infon key="updated_at">2023-09-19T09:09:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10952" length="4"/>
<text>S198</text>
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<annotation id="278">
<infon key="score">0.99713284</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:05:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10970" length="18"/>
<text>polar interactions</text>
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<annotation id="279">
<infon key="score">0.9994918</infon>
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<infon key="updated_at">2023-09-19T09:09:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11012" length="4"/>
<text>N140</text>
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<annotation id="280">
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<infon key="updated_at">2023-09-19T09:09:38Z</infon>
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<location offset="11018" length="4"/>
<text>S163</text>
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<annotation id="281">
<infon key="score">0.9995003</infon>
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<infon key="updated_at">2023-09-19T09:09:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11028" length="4"/>
<text>Y165</text>
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<annotation id="282">
<infon key="score">0.9794397</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11048" length="4"/>
<text>KRM1</text>
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<annotation id="283">
<infon key="score">0.9987478</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11057" length="4"/>
<text>DKK1</text>
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<annotation id="284">
<infon key="score">0.9995042</infon>
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<location offset="11084" length="4"/>
<text>W206</text>
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<annotation id="285">
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<infon key="updated_at">2023-09-19T09:09:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11090" length="4"/>
<text>L190</text>
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<infon key="updated_at">2023-09-19T09:09:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11100" length="4"/>
<text>C189</text>
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<annotation id="287">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11136" length="4"/>
<text>DKK1</text>
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<annotation id="288">
<infon key="score">0.9995389</infon>
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<infon key="updated_at">2023-09-19T09:10:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11141" length="4"/>
<text>R224</text>
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<annotation id="289">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11149" length="15"/>
<text>hydrogen bonded</text>
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<annotation id="290">
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<infon key="type">residue_name_number</infon>
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<infon key="updated_at">2023-09-19T09:10:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11168" length="4"/>
<text>Y105</text>
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<annotation id="291">
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<infon key="updated_at">2023-09-19T09:08:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11177" length="4"/>
<text>W106</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11185" length="4"/>
<text>KRM1</text>
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<annotation id="293">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11211" length="3"/>
<text>Dkk</text>
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<annotation id="294">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11215" length="25"/>
<text>charge reversal mutations</text>
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<infon key="score">0.9982382</infon>
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<infon key="updated_at">2023-09-19T09:08:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11258" length="6"/>
<text>murine</text>
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<annotation id="583">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:10:24Z</infon>
<location offset="11298" length="3"/>
<text>Krm</text>
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<annotation id="296">
<infon key="score">0.9989784</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:10:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11310" length="5"/>
<text>K211E</text>
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<annotation id="297">
<infon key="score">0.9990165</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:10:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11320" length="5"/>
<text>R203E</text>
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<annotation id="298">
<infon key="score">0.9978951</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:08:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11327" length="5"/>
<text>mouse</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:10:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11333" length="5"/>
<text>K217E</text>
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<annotation id="300">
<infon key="score">0.9990754</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:10:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11343" length="5"/>
<text>R209E</text>
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<annotation id="301">
<infon key="score">0.99816555</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11395" length="3"/>
<text>Dkk</text>
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<annotation id="302">
<infon key="score">0.99893934</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11430" length="17"/>
<text>DKK1 binding site</text>
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<annotation id="303">
<infon key="score">0.9929836</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11451" length="4"/>
<text>KRM1</text>
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<annotation id="304">
<infon key="score">0.4963542</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11459" length="11"/>
<text>introducing</text>
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<infon key="score">0.9917952</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:51:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11471" length="19"/>
<text>glycosylation sites</text>
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<infon key="score">0.9976114</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11498" length="2"/>
<text>KR</text>
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<annotation id="307">
<infon key="score">0.6751963</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:07:36Z</infon>
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<location offset="11502" length="11"/>
<text>90DVS92→NVS</text>
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<annotation id="308">
<infon key="score">0.9923799</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:07:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11519" length="3"/>
<text>WSC</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:11:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11524" length="13"/>
<text>189VCF191→NCS</text>
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<infon key="score">0.99895453</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:08:02Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11563" length="3"/>
<text>DKK</text>
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<annotation id="311">
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<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:11:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11604" length="16"/>
<text>N-linked glycans</text>
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<infon key="score">0.9990034</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11624" length="29"/>
<text>protein-protein-binding sites</text>
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<annotation id="313">
<infon key="score">0.9990411</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11690" length="26"/>
<text>protein-binding interfaces</text>
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<infon key="score">0.88388306</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11723" length="10"/>
<text>ectodomain</text>
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<infon key="updated_at">2023-09-19T10:00:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11734" length="7"/>
<text>mutants</text>
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<infon key="score">0.99901456</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11776" length="9"/>
<text>wild-type</text>
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<infon key="score">0.99868995</infon>
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<infon key="updated_at">2023-09-19T09:04:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11863" length="3"/>
<text>SPR</text>
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<infon key="score">0.998916</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11873" length="11"/>
<text>full-length</text>
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<infon key="score">0.9990551</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11885" length="4"/>
<text>DKK1</text>
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<infon key="score">0.98682505</infon>
<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-19T09:12:34Z</infon>
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<text>mutant</text>
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<infon key="identifier">MESH:</infon>
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<infon key="updated_at">2023-09-19T09:12:08Z</infon>
<location offset="11947" length="8"/>
<text>N-glycan</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:06:42Z</infon>
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<location offset="11968" length="9"/>
<text>interface</text>
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<infon key="score">0.9992186</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:18:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11985" length="3"/>
<text>CUB</text>
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<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11997" length="13"/>
<text>309NQA311→NQS</text>
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<infon key="score">0.9991409</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12017" length="9"/>
<text>wild-type</text>
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<infon key="score">0.9986671</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12035" length="4"/>
<text>DKK1</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>12076</offset>
<text>Identification of a Direct LRP6-KRM1 Binding Site</text>
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<infon key="score">0.9978304</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:46Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12103" length="22"/>
<text>LRP6-KRM1 Binding Site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12126</offset>
<text>The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).</text>
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<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:01Z</infon>
<infon key="identifier">GO:</infon>
<location offset="12130" length="27"/>
<text>LRP6PE3PE4-DKK1CRD2-KRM1ECD</text>
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12166" length="9"/>
<text>structure</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12215" length="4"/>
<text>KRM1</text>
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<annotation id="330">
<infon key="score">0.99859744</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12224" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="501">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:15:14Z</infon>
<location offset="12265" length="12"/>
<text>complex with</text>
</annotation>
<annotation id="331">
<infon key="score">0.9976246</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12289" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="332">
<infon key="score">0.999175</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12301" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="333">
<infon key="score">0.99937844</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12306" length="10"/>
<text>ectodomain</text>
</annotation>
<annotation id="587">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:50Z</infon>
<location offset="12318" length="12"/>
<text>PE1PE2PE3PE4</text>
</annotation>
<annotation id="588">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:01Z</infon>
<location offset="12331" length="10"/>
<text>horse shoe</text>
</annotation>
<annotation id="334">
<infon key="score">0.99863327</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12385" length="19"/>
<text>electron microscopy</text>
</annotation>
<annotation id="335">
<infon key="score">0.9984401</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12406" length="2"/>
<text>EM</text>
</annotation>
<annotation id="336">
<infon key="score">0.99649286</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12413" length="28"/>
<text>small-angle X-ray scattering</text>
</annotation>
<annotation id="337">
<infon key="score">0.4334478</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12461" length="6"/>
<text>PE3PE4</text>
</annotation>
<annotation id="338">
<infon key="score">0.99877995</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12481" length="12"/>
<text>superimposed</text>
</annotation>
<annotation id="339">
<infon key="score">0.99630725</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12498" length="3"/>
<text>PE4</text>
</annotation>
<annotation id="340">
<infon key="score">0.99660873</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12507" length="3"/>
<text>PE3</text>
</annotation>
<annotation id="341">
<infon key="score">0.99758327</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:08:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12518" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="589">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:17Z</infon>
<location offset="12545" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="590">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:30Z</infon>
<location offset="12549" length="6"/>
<text>PE1PE2</text>
</annotation>
<annotation id="342">
<infon key="score">0.9973882</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12556" length="9"/>
<text>structure</text>
</annotation>
<annotation id="343">
<infon key="score">0.9988292</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12570" length="12"/>
<text>superimposed</text>
</annotation>
<annotation id="344">
<infon key="score">0.99678135</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12587" length="3"/>
<text>PE2</text>
</annotation>
<annotation id="345">
<infon key="score">0.99711955</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12596" length="3"/>
<text>PE3</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12642</offset>
<text>For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.</text>
<annotation id="346">
<infon key="score">0.99889165</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12740" length="19"/>
<text>Ca2+-binding region</text>
</annotation>
<annotation id="347">
<infon key="score">0.70204115</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12763" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="594">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:10Z</infon>
<location offset="12792" length="3"/>
<text>PE2</text>
</annotation>
<annotation id="595">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:23Z</infon>
<location offset="12796" length="11"/>
<text>β propeller</text>
</annotation>
<annotation id="348">
<infon key="score">0.99911326</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12811" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="667">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:04:32Z</infon>
<location offset="12821" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="349">
<infon key="score">0.9994591</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12846" length="4"/>
<text>R307</text>
</annotation>
<annotation id="350">
<infon key="score">0.99941707</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12852" length="4"/>
<text>I308</text>
</annotation>
<annotation id="351">
<infon key="score">0.99950254</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12862" length="4"/>
<text>N309</text>
</annotation>
<annotation id="591">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:38Z</infon>
<location offset="12882" length="30"/>
<text>Ca2+-binding β connection loop</text>
</annotation>
<annotation id="352">
<infon key="score">0.8115768</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12916" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="353">
<infon key="score">0.998543</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13010" length="12"/>
<text>binding site</text>
</annotation>
<annotation id="354">
<infon key="score">0.99863666</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13026" length="12"/>
<text>β propellers</text>
</annotation>
<annotation id="355">
<infon key="score">0.99799347</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13060" length="8"/>
<text>arginine</text>
</annotation>
<annotation id="356">
<infon key="score">0.997482</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13069" length="6"/>
<text>lysine</text>
</annotation>
<annotation id="357">
<infon key="score">0.99793506</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13077" length="10"/>
<text>isoleucine</text>
</annotation>
<annotation id="358">
<infon key="score">0.99770665</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:15:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13093" length="10"/>
<text>asparagine</text>
</annotation>
<annotation id="359">
<infon key="score">0.9350921</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:07:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13124" length="13"/>
<text>N-X-I-(G)-R/K</text>
</annotation>
<annotation id="360">
<infon key="score">0.99911493</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13160" length="4"/>
<text>DKK1</text>
</annotation>
<annotation id="361">
<infon key="score">0.999201</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:16:15Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13169" length="4"/>
<text>SOST</text>
</annotation>
<annotation id="362">
<infon key="score">0.99911827</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13185" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="363">
<infon key="score">0.9990943</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:58:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13201" length="11"/>
<text>propeller 1</text>
</annotation>
<annotation id="596">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:38Z</infon>
<location offset="13257" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="597">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:48Z</infon>
<location offset="13261" length="3"/>
<text>CUB</text>
</annotation>
<annotation id="665">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:00:12Z</infon>
<location offset="13265" length="8"/>
<text>binds to</text>
</annotation>
<annotation id="598">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:26:02Z</infon>
<location offset="13274" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="599">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:26:14Z</infon>
<location offset="13278" length="3"/>
<text>PE2</text>
</annotation>
<annotation id="364">
<infon key="score">0.99864656</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13291" length="3"/>
<text>SPR</text>
</annotation>
<annotation id="365">
<infon key="score">0.99910635</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13323" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="366">
<infon key="score">0.9700352</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:27:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13341" length="15"/>
<text>GlycoCUB mutant</text>
</annotation>
<annotation id="600">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:26:33Z</infon>
<location offset="13360" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="601">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:26:50Z</infon>
<location offset="13364" length="3"/>
<text>ECD</text>
</annotation>
<annotation id="367">
<infon key="score">0.9965527</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:16:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13380" length="20"/>
<text>N-glycosylation site</text>
</annotation>
<annotation id="368">
<infon key="score">0.9995345</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:14:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13404" length="4"/>
<text>N309</text>
</annotation>
<annotation id="602">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:27:18Z</infon>
<location offset="13426" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="603">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:27:28Z</infon>
<location offset="13430" length="6"/>
<text>PE1PE2</text>
</annotation>
<annotation id="369">
<infon key="score">0.9990541</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:24:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13476" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="370">
<infon key="score">0.9986665</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13487" length="3"/>
<text>Dkk</text>
</annotation>
<annotation id="604">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:27:42Z</infon>
<location offset="13492" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="605">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:27:53Z</infon>
<location offset="13496" length="3"/>
<text>ECD</text>
</annotation>
<annotation id="371">
<infon key="score">0.9928445</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:01:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13500" length="5"/>
<text>bound</text>
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<annotation id="372">
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:01:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13533" length="2"/>
<text>to</text>
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<annotation id="606">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:28:11Z</infon>
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<text>LRP6</text>
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<annotation id="607">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:28:21Z</infon>
<location offset="13540" length="6"/>
<text>PE1PE2</text>
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<annotation id="373">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
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<location offset="13615" length="4"/>
<text>KRM1</text>
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<annotation id="608">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:28:36Z</infon>
<location offset="13624" length="4"/>
<text>LRP6</text>
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<annotation id="609">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:28:47Z</infon>
<location offset="13628" length="6"/>
<text>PE3PE4</text>
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<annotation id="374">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13636" length="15"/>
<text>Introduction of</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:24:25Z</infon>
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<location offset="13655" length="20"/>
<text>N-glycosylation site</text>
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<location offset="13679" length="4"/>
<text>N309</text>
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<annotation id="610">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
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<text>KRM1</text>
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<annotation id="611">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:29:13Z</infon>
<location offset="13691" length="3"/>
<text>ECD</text>
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<annotation id="614">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:29:54Z</infon>
<location offset="13705" length="4"/>
<text>LRP6</text>
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<annotation id="615">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:30:05Z</infon>
<location offset="13709" length="6"/>
<text>PE1PE2</text>
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<annotation id="377">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
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<annotation id="378">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:06:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13818" length="12"/>
<text>binding site</text>
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<annotation id="612">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
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<text>KRM1</text>
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<annotation id="613">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="13843" length="3"/>
<text>CUB</text>
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<annotation id="616">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
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<infon key="updated_at">2023-09-19T09:30:19Z</infon>
<location offset="13851" length="4"/>
<text>LRP6</text>
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<annotation id="617">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:30:28Z</infon>
<location offset="13855" length="3"/>
<text>PE2</text>
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<annotation id="379">
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<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:23:58Z</infon>
<infon key="identifier">GO:</infon>
<location offset="13906" length="8"/>
<text>Lrp6-Krm</text>
</annotation>
<annotation id="523">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="13957" length="3"/>
<text>Wnt</text>
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<annotation id="380">
<infon key="score">0.9983664</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13991" length="4"/>
<text>Lrp6</text>
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<annotation id="676">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:07:56Z</infon>
<location offset="14072" length="12"/>
<text>binding site</text>
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<annotation id="618">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:30:44Z</infon>
<location offset="14095" length="4"/>
<text>LRP6</text>
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<annotation id="619">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:30:55Z</infon>
<location offset="14099" length="3"/>
<text>PE1</text>
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<annotation id="381">
<infon key="score">0.9989984</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:08:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14194" length="16"/>
<text>Krm binding site</text>
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<annotation id="382">
<infon key="score">0.9991382</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:31:01Z</infon>
<infon key="identifier">GO:</infon>
<location offset="14238" length="8"/>
<text>LRP6-Krm</text>
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<annotation id="383">
<infon key="score">0.99640095</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:34:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14267" length="11"/>
<text>presence of</text>
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<annotation id="384">
<infon key="score">0.998844</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:08:02Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14279" length="3"/>
<text>DKK</text>
</annotation>
<annotation id="385">
<infon key="score">0.9993789</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:58:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14300" length="9"/>
<text>propeller</text>
</annotation>
<annotation id="386">
<infon key="score">0.99949026</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:31:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14311" length="3"/>
<text>PE1</text>
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<annotation id="387">
<infon key="score">0.99948514</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14322" length="3"/>
<text>PE2</text>
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<annotation id="388">
<infon key="score">0.99907756</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14330" length="4"/>
<text>LRP6</text>
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<annotation id="389">
<infon key="score">0.66727495</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:58:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14352" length="3"/>
<text>Krm</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>14374</offset>
<text>Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.</text>
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:08:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14411" length="25"/>
<text>KRM1CUB-LRP6PE2 interface</text>
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<infon key="score">0.9994892</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:59:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14467" length="3"/>
<text>Krm</text>
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<annotation id="620">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:32:33Z</infon>
<location offset="14495" length="4"/>
<text>LRP6</text>
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<annotation id="621">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:32:43Z</infon>
<location offset="14499" length="3"/>
<text>PE3</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:32:57Z</infon>
<location offset="14507" length="4"/>
<text>DKK1</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:06Z</infon>
<location offset="14511" length="4"/>
<text>CRD2</text>
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<infon key="score">0.9989036</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:32:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14544" length="17"/>
<text>negative-stain EM</text>
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<infon key="score">0.9989684</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:13:24Z</infon>
<infon key="identifier">MESH:</infon>
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<text>small-angle X-ray scattering</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:25Z</infon>
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<text>LRP6</text>
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<annotation id="625">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:35Z</infon>
<location offset="14610" length="12"/>
<text>PE1PE2PE3PE4</text>
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<annotation id="394">
<infon key="score">0.9033568</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:31:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14624" length="12"/>
<text>in isolation</text>
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<annotation id="395">
<infon key="score">0.99816304</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:31:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14641" length="15"/>
<text>in complex with</text>
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<infon key="score">0.9985501</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14657" length="4"/>
<text>Dkk1</text>
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<infon key="score">0.99890447</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:32:16Z</infon>
<infon key="identifier">MESH:</infon>
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<text>negative-stain EM</text>
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<annotation id="398">
<infon key="score">0.9991421</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14689" length="11"/>
<text>full-length</text>
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<infon key="score">0.999263</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14701" length="4"/>
<text>LRP6</text>
</annotation>
<annotation id="400">
<infon key="score">0.99935347</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14706" length="10"/>
<text>ectodomain</text>
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<infon key="score">0.99760216</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:01:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14733" length="6"/>
<text>curved</text>
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<infon key="score">0.9706499</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:01:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14741" length="13"/>
<text>platform-like</text>
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<annotation id="403">
<infon key="score">0.99944097</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:25:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14808" length="3"/>
<text>PE2</text>
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<infon key="score">0.9994288</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14816" length="3"/>
<text>PE3</text>
</annotation>
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<infon key="score">0.9995103</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:31:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14868" length="3"/>
<text>Krm</text>
</annotation>
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<infon key="score">0.5479226</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14876" length="3"/>
<text>Dkk</text>
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<infon key="score">0.9992786</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14911" length="4"/>
<text>LRP6</text>
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<annotation id="408">
<infon key="score">0.9993906</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14916" length="10"/>
<text>ectodomain</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15012</offset>
<text>Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.</text>
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<infon key="score">0.9987791</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:43Z</infon>
<infon key="identifier">MESH:</infon>
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<text>structural and biophysical studies</text>
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
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<text>Wnt</text>
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<infon key="score">0.99944466</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:05:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15161" length="10"/>
<text>ectodomain</text>
</annotation>
<annotation id="411">
<infon key="score">0.98785317</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15172" length="9"/>
<text>structure</text>
</annotation>
<annotation id="525">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="15194" length="3"/>
<text>Wnt</text>
</annotation>
<annotation id="412">
<infon key="score">0.99765694</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15208" length="4"/>
<text>Krm1</text>
</annotation>
<annotation id="413">
<infon key="score">0.9876721</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15308" length="4"/>
<text>KRM1</text>
</annotation>
<annotation id="626">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:34:21Z</infon>
<location offset="15362" length="7"/>
<text>Krm-Dkk</text>
</annotation>
<annotation id="414">
<infon key="score">0.9881404</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:34:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15424" length="6"/>
<text>Lrp5/6</text>
</annotation>
<annotation id="415">
<infon key="score">0.9984548</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15432" length="3"/>
<text>Dkk</text>
</annotation>
<annotation id="416">
<infon key="score">0.9982216</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:34:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15441" length="3"/>
<text>Krm</text>
</annotation>
<annotation id="417">
<infon key="score">0.9973328</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:08:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15471" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="418">
<infon key="score">0.9021877</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:33:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15500" length="34"/>
<text>in silico and biophysical analyses</text>
</annotation>
<annotation id="419">
<infon key="score">0.97991484</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:34:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15555" length="26"/>
<text>LRP6-KRM1 interaction site</text>
</annotation>
<annotation id="420">
<infon key="score">0.833017</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:34:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15719" length="3"/>
<text>Krm</text>
</annotation>
<annotation id="421">
<infon key="score">0.9991188</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:24:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15738" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="422">
<infon key="score">0.9983999</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:10:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15749" length="3"/>
<text>Dkk</text>
</annotation>
<annotation id="526">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:16:32Z</infon>
<location offset="15768" length="3"/>
<text>Wnt</text>
</annotation>
<annotation id="532">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:17:09Z</infon>
<location offset="15772" length="11"/>
<text>co-receptor</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>15806</offset>
<text>Experimental Procedures</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>15830</offset>
<text>Large-Scale Mammalian Expression and Protein Purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>15888</offset>
<text>KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>17365</offset>
<text>Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>17945</offset>
<text>To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>18220</offset>
<text>Crystallization and Data Collection</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>18256</offset>
<text>All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>20188</offset>
<text>Structure Determination</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>20212</offset>
<text>Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>20748</offset>
<text>For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (<I/σI> = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of >10 and log likelihood gains (LLG) of >200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>21945</offset>
<text>We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>22631</offset>
<text>Surface Plasmon Resonance</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>22657</offset>
<text>Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">title_1</infon>
<offset>22822</offset>
<text>Author Contributions</text>
</passage>
<passage>
<infon key="section_type">AUTH_CONT</infon>
<infon key="type">paragraph</infon>
<offset>22843</offset>
<text>M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. M.Z. and E.Y.J. designed the research. M.Z. wrote the paper with input from all other authors.</text>
</passage>
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<text>References</text>
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<text>Crystallographic evidence for a domain motion in rat nucleoside triphosphate diphosphohydrolase (NTPDase) 1</text>
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<text>Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin</text>
</passage>
<passage>
<infon key="section_type">KEYWORD</infon>
<infon key="type">title_1</infon>
<offset>26731</offset>
<text>Accession Numbers</text>
</passage>
<passage>
<infon key="section_type">KEYWORD</infon>
<infon key="type">paragraph</infon>
<offset>26749</offset>
<text>Coordinates and structure factors have been deposited in the PDB with succession numbers PDB: 5FWS, 5FWT, 5FWU, 5FWV, and 5FWW.</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>26877</offset>
<text>Supplemental Information</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>26902</offset>
<text>Supplemental Information includes one figure and can be found with this article online at http://dx.doi.org/10.1016/j.str.2016.06.020.</text>
</passage>
<passage>
<infon key="file">gr1.jpg</infon>
<infon key="id">fig1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27037</offset>
<text>Structure of Unliganded KRM1ECD</text>
<annotation id="423">
<infon key="score">0.99732375</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:57:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27037" length="9"/>
<text>Structure</text>
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<infon key="updated_at">2023-09-19T09:42:59Z</infon>
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<text>Unliganded</text>
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<location offset="27061" length="4"/>
<text>KRM1</text>
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<annotation id="628">
<infon key="type">structure_element</infon>
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<infon key="annotator">cleaner0</infon>
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<text>ECD</text>
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<passage>
<infon key="file">gr1.jpg</infon>
<infon key="id">fig1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27069</offset>
<text>(A) The KRM1ECD fold (crystal form I) colored blue to red from the N to C terminus. Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.</text>
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<location offset="27077" length="4"/>
<text>KRM1</text>
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<text>ECD</text>
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<text>crystal form I</text>
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<infon key="identifier">SO:</infon>
<location offset="27153" length="9"/>
<text>Cysteines</text>
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<annotation id="426">
<infon key="score">0.9977498</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:51:54Z</infon>
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<location offset="27183" length="19"/>
<text>glycosylation sites</text>
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<annotation id="427">
<infon key="score">0.9988972</infon>
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<infon key="identifier">CHEBI:</infon>
<location offset="27224" length="7"/>
<text>calcium</text>
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<annotation id="428">
<infon key="score">0.9986633</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:54:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27275" length="5"/>
<text>F207S</text>
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<annotation id="429">
<infon key="score">0.99778545</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:44:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27330" length="6"/>
<text>humans</text>
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</passage>
<passage>
<infon key="file">gr1.jpg</infon>
<infon key="id">fig1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27356</offset>
<text>(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).</text>
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<infon key="score">0.99862194</infon>
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<infon key="updated_at">2023-09-19T08:53:58Z</infon>
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<location offset="27360" length="13"/>
<text>Superposition</text>
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<text>KRM1</text>
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<infon key="annotator">cleaner0</infon>
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<text>ECD</text>
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<passage>
<infon key="file">gr1.jpg</infon>
<infon key="id">fig1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27486</offset>
<text>(C) Superposition of KRM1ECD from the three crystal forms. Alignment scores for each pairing are indicated on the dashed triangle.</text>
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<infon key="score">0.9977836</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:53:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27490" length="13"/>
<text>Superposition</text>
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<annotation id="634">
<infon key="type">protein</infon>
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<infon key="annotator">cleaner0</infon>
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<location offset="27507" length="4"/>
<text>KRM1</text>
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<annotation id="635">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:45:14Z</infon>
<location offset="27511" length="3"/>
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<annotation id="636">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:45:30Z</infon>
<location offset="27530" length="13"/>
<text>crystal forms</text>
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<annotation id="432">
<infon key="score">0.9983538</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:45:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27545" length="16"/>
<text>Alignment scores</text>
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</passage>
<passage>
<infon key="file">gr2.jpg</infon>
<infon key="id">fig2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27617</offset>
<text>Insight into Ternary Complex Formation</text>
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<passage>
<infon key="file">gr2.jpg</infon>
<infon key="id">fig2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27656</offset>
<text>(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D).</text>
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<infon key="score">0.9669953</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:45:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27664" length="9"/>
<text>structure</text>
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<annotation id="434">
<infon key="score">0.99905986</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:45:01Z</infon>
<infon key="identifier">GO:</infon>
<location offset="27689" length="27"/>
<text>LRP6PE3PE4-DKK1CRD2-KRM1ECD</text>
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<annotation id="435">
<infon key="score">0.99864715</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27726" length="4"/>
<text>DKK1</text>
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<annotation id="593">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:18:50Z</infon>
<location offset="27766" length="3"/>
<text>PE3</text>
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<annotation id="436">
<infon key="score">0.9989537</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:04:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27780" length="4"/>
<text>LRP6</text>
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<annotation id="637">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:46:15Z</infon>
<location offset="27800" length="6"/>
<text>KR-WSC</text>
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<annotation id="437">
<infon key="score">0.8255878</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T15:06:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27822" length="4"/>
<text>KRM1</text>
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<annotation id="638">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:46:51Z</infon>
<location offset="27872" length="25"/>
<text>N-glycan attachment sites</text>
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<passage>
<infon key="file">gr2.jpg</infon>
<infon key="id">fig2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>27907</offset>
<text>(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.</text>
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<infon key="score">0.99875593</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:04:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27911" length="3"/>
<text>SPR</text>
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<annotation id="439">
<infon key="score">0.999115</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:48:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27941" length="11"/>
<text>full-length</text>
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<annotation id="440">
<infon key="score">0.9985942</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27953" length="4"/>
<text>DKK1</text>
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<annotation id="441">
<infon key="score">0.9968902</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T10:03:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27962" length="12"/>
<text>SUMO fusions</text>
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<annotation id="442">
<infon key="score">0.9980323</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T08:56:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27978" length="4"/>
<text>DKK1</text>
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<annotation id="443">
<infon key="score">0.9991273</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:12:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28022" length="9"/>
<text>wild-type</text>
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<annotation id="639">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:47:07Z</infon>
<location offset="28032" length="4"/>
<text>KRM1</text>
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<annotation id="640">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:47:17Z</infon>
<location offset="28036" length="3"/>
<text>ECD</text>
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<passage>
<infon key="file">gr2.jpg</infon>
<infon key="id">fig2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>28041</offset>
<text>(C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1.</text>
<annotation id="444">
<infon key="score">0.9989432</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:47:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28066" length="26"/>
<text>DKK1CRD2-KRM1ECD interface</text>
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<annotation id="445">
<infon key="score">0.9983902</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-19T09:47:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28115" length="9"/>
<text>interface</text>
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<text>(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A).</text>
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<text>(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.</text>
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<text>(B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.</text>
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<text>(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.</text>
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<text>(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation.</text>
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<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead><tr><th/><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>LRP6<sub>PE3PE4</sub>-DKK<sub>CRD2</sub>-KRM1<sub>ECD</sub></th></tr></thead><tbody><tr><td>Crystal form</td><td>I</td><td>I</td><td>II</td><td>III</td><td>I</td></tr><tr><td>X-ray source</td><td>Diamond i04</td><td>Diamond i03</td><td>Diamond i03</td><td>Diamond i04</td><td>Diamond i04</td></tr><tr><td>Wavelength (Å)</td><td>0.9793</td><td>0.9700</td><td>0.9700</td><td>0.9795</td><td>0.9795</td></tr><tr><td>Space group</td><td><italic>P</italic>3<sub>1</sub>21</td><td><italic>P</italic>3<sub>1</sub>21</td><td><italic>P</italic>4<sub>3</sub></td><td><italic>P</italic>4<sub>1</sub>2<sub>1</sub>2</td><td><italic>C</italic>222<sub>1</sub></td></tr><tr><td>Unit cell a/α (Å/°)</td><td>50.9/90</td><td>50.5/90</td><td>65.8/90</td><td>67.8/90</td><td>86.9/90</td></tr><tr><td>b/β (Å/°)</td><td>50.9/90</td><td>50.5/90</td><td>65.8/90</td><td>67.8/90</td><td>100.1/90</td></tr><tr><td>c/γ (Å/°)</td><td>188.4/120</td><td>187.4/120</td><td>75.0/90</td><td>198.2/90</td><td>270.7/90</td></tr><tr><td>Wilson B factor (Å<sup>2</sup>)</td><td>31</td><td>41</td><td>76</td><td>77</td><td>NA</td></tr><tr><td>Resolution range (Å)</td><td>47.10–1.90 (1.95–1.90)</td><td>62.47–2.10 (2.16–2.10)</td><td>75.00–2.80 (2.99–2.80)</td><td>67.80–3.20 (3.42–3.20)</td><td>67.68–3.50 (7.16–6.40, 3.92–3.50)</td></tr><tr><td>Unique reflections</td><td>23,300 (1,524)</td><td>17,089 (1,428)</td><td>7,964 (1,448)</td><td>8,171 (1,343)</td><td>8,070 (723, 645)</td></tr><tr><td>Average multiplicity</td><td>9.1 (9.2)</td><td>5.2 (5.3)</td><td>3.7 (3.7)</td><td>22.7 (12.6)</td><td>3.8 (3.5, 4.4)</td></tr><tr><td>Completeness (%)</td><td>99.8 (98.5)</td><td>100 (100)</td><td>99.8 (100)</td><td>98.8 (93.4)</td><td>51.6 (98.5, 14.1)</td></tr><tr><td>&lt;<italic>I</italic>/<italic>σI</italic>&gt;</td><td>11.4 (1.7)</td><td>12.0 (1.7)</td><td>14.9 (1.5)</td><td>13.1 (1.9)</td><td>4.6 (4.1, 2.2)</td></tr><tr><td><italic>R</italic><sub>merge</sub> (%)</td><td>14.8 (158.3)</td><td>9.3 (98.0)</td><td>6.2 (98.9)</td><td>29.8 (142.2)</td><td>44.9 (40.5, 114.2)</td></tr><tr><td><italic>R</italic><sub>pim</sub> (%)</td><td>15.7 (55.3)</td><td>10.3 (109.0)</td><td>3.7 (53.8)</td><td>6.3 (40.0)</td><td>24.7 (23.9, 59.9)</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Refinement</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td><italic>R</italic><sub>work</sub> (%)</td><td>17.9</td><td>18.4</td><td>21.6</td><td>20.2</td><td>32.1</td></tr><tr><td><italic>R</italic><sub>free</sub> (%)</td><td>22.7</td><td>23.2</td><td>30.7</td><td>27.1</td><td>35.5</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>No. of Non-Hydrogen Atoms</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Protein</td><td>2,260</td><td>2,301</td><td>2,102</td><td>2,305</td><td>7,730</td></tr><tr><td>N-glycans</td><td>42</td><td>42</td><td>28</td><td>28</td><td>0</td></tr><tr><td>Water</td><td>79</td><td>54</td><td>0</td><td>2</td><td>0</td></tr><tr><td>Ligands</td><td>6</td><td>6</td><td>2</td><td>5</td><td>0</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Average B factor (Å<sup>2</sup>)</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Protein</td><td>63</td><td>65</td><td>108</td><td>84</td><td>–</td></tr><tr><td>N-glycans</td><td>35</td><td>46</td><td>102</td><td>18</td><td>–</td></tr><tr><td>Water</td><td>68</td><td>85</td><td>–</td><td>75</td><td>–</td></tr><tr><td>Ligands</td><td>36</td><td>47</td><td>91</td><td>75</td><td>66</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>RMSD from Ideality</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Bond lengths (Å)</td><td>0.020</td><td>0.016</td><td>0.019</td><td>0.016</td><td>0.004</td></tr><tr><td>Bond angles (°)</td><td>2.050</td><td>1.748</td><td>1.952</td><td>1.796</td><td>0.770</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Ramachandran Plot</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Favored (%)</td><td>96.8</td><td>95.5</td><td>96.9</td><td>94.9</td><td>92.3</td></tr><tr><td>Allowed (%)</td><td>99.7</td><td>100.0</td><td>100.0</td><td>99.7</td><td>99.8</td></tr><tr><td>Number of outliers</td><td>1</td><td>0</td><td>0</td><td>1</td><td>2</td></tr><tr><td>PDB code</td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWS" id="intref0085">5FWS</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWT" id="intref0090">5FWT</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWU" id="intref0095">5FWU</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWV" id="intref0100">5FWV</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWW" id="intref0105">5FWW</ext-link></td></tr></tbody></table>
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<text> KRM1ECD KRM1ECD KRM1ECD KRM1ECD LRP6PE3PE4-DKKCRD2-KRM1ECD Crystal form I I II III I X-ray source Diamond i04 Diamond i03 Diamond i03 Diamond i04 Diamond i04 Wavelength (Å) 0.9793 0.9700 0.9700 0.9795 0.9795 Space group P3121 P3121 P43 P41212 C2221 Unit cell a/α (Å/°) 50.9/90 50.5/90 65.8/90 67.8/90 86.9/90 b/β (Å/°) 50.9/90 50.5/90 65.8/90 67.8/90 100.1/90 c/γ (Å/°) 188.4/120 187.4/120 75.0/90 198.2/90 270.7/90 Wilson B factor (Å2) 31 41 76 77 NA Resolution range (Å) 47.10–1.90 (1.95–1.90) 62.47–2.10 (2.16–2.10) 75.00–2.80 (2.99–2.80) 67.80–3.20 (3.42–3.20) 67.68–3.50 (7.16–6.40, 3.92–3.50) Unique reflections 23,300 (1,524) 17,089 (1,428) 7,964 (1,448) 8,171 (1,343) 8,070 (723, 645) Average multiplicity 9.1 (9.2) 5.2 (5.3) 3.7 (3.7) 22.7 (12.6) 3.8 (3.5, 4.4) Completeness (%) 99.8 (98.5) 100 (100) 99.8 (100) 98.8 (93.4) 51.6 (98.5, 14.1) <I/σI> 11.4 (1.7) 12.0 (1.7) 14.9 (1.5) 13.1 (1.9) 4.6 (4.1, 2.2) Rmerge (%) 14.8 (158.3) 9.3 (98.0) 6.2 (98.9) 29.8 (142.2) 44.9 (40.5, 114.2) Rpim (%) 15.7 (55.3) 10.3 (109.0) 3.7 (53.8) 6.3 (40.0) 24.7 (23.9, 59.9) Refinement Rwork (%) 17.9 18.4 21.6 20.2 32.1 Rfree (%) 22.7 23.2 30.7 27.1 35.5 No. of Non-Hydrogen Atoms Protein 2,260 2,301 2,102 2,305 7,730 N-glycans 42 42 28 28 0 Water 79 54 0 2 0 Ligands 6 6 2 5 0 Average B factor (Å2) Protein 63 65 108 84 – N-glycans 35 46 102 18 – Water 68 85 – 75 – Ligands 36 47 91 75 66 RMSD from Ideality Bond lengths (Å) 0.020 0.016 0.019 0.016 0.004 Bond angles (°) 2.050 1.748 1.952 1.796 0.770 Ramachandran Plot Favored (%) 96.8 95.5 96.9 94.9 92.3 Allowed (%) 99.7 100.0 100.0 99.7 99.8 Number of outliers 1 0 0 1 2 PDB code 5FWS 5FWT 5FWU 5FWV 5FWW </text>
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<text>KRM1</text>
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<text>KRM1</text>
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<text>LRP6PE3PE4-DKKCRD2-KRM1ECD</text>
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<text>Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not announced.</text>
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<text>diffraction data</text>
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<!DOCTYPE collection SYSTEM "BioC.dtd">
<collection>
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<id>5063996</id>
<infon key="license">CC BY</infon>
<infon key="tt_curatable">no</infon>
<infon key="tt_version">2</infon>
<infon key="tt_round">2</infon>
<passage>
<infon key="alt-title">Mechanism of Arabinoxylanase</infon>
<infon key="article-id_doi">10.1074/jbc.M116.743948</infon>
<infon key="article-id_pmc">5063996</infon>
<infon key="article-id_pmid">27531750</infon>
<infon key="article-id_publisher-id">M116.743948</infon>
<infon key="fpage">22149</infon>
<infon key="issue">42</infon>
<infon key="kwd">cellulosome crystallography enzyme kinetics enzyme mechanism glycoside hydrolase</infon>
<infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon>
<infon key="lpage">22159</infon>
<infon key="name_0">surname:Labourel;given-names:Aurore</infon>
<infon key="name_1">surname:Crouch;given-names:Lucy I.</infon>
<infon key="name_10">surname:Najmudin;given-names:Shabir</infon>
<infon key="name_11">surname:Baslé;given-names:Arnaud</infon>
<infon key="name_12">surname:Cuskin;given-names:Fiona</infon>
<infon key="name_2">surname:Brás;given-names:Joana L. A.</infon>
<infon key="name_3">surname:Jackson;given-names:Adam</infon>
<infon key="name_4">surname:Rogowski;given-names:Artur</infon>
<infon key="name_5">surname:Gray;given-names:Joseph</infon>
<infon key="name_6">surname:Yadav;given-names:Madhav P.</infon>
<infon key="name_7">surname:Henrissat;given-names:Bernard</infon>
<infon key="name_8">surname:Fontes;given-names:Carlos M. G. A.</infon>
<infon key="name_9">surname:Gilbert;given-names:Harry J.</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">291</infon>
<infon key="year">2016</infon>
<offset>0</offset>
<text>The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*</text>
<annotation id="1">
<infon key="score">0.99930334</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23" length="16"/>
<text>Arabinoxylanases</text>
</annotation>
<annotation id="925">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:47:12Z</infon>
<location offset="54" length="16"/>
<text>Highly Decorated</text>
</annotation>
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<infon key="score">0.9991499</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:31Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="71" length="6"/>
<text>Xylans</text>
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<passage>
<infon key="section_type">ABSTRACT</infon>
<infon key="type">abstract</infon>
<offset>79</offset>
<text>The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.</text>
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<infon key="score">0.9984566</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:41Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>plant</text>
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<infon key="score">0.99672145</infon>
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<infon key="updated_at">2023-09-18T10:24:48Z</infon>
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<text>Xylan</text>
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<infon key="updated_at">2023-09-18T10:24:41Z</infon>
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<text>plant</text>
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<infon key="score">0.9991608</infon>
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<infon key="updated_at">2023-09-18T10:24:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="292" length="12"/>
<text>β-1,4-xylose</text>
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<infon key="score">0.99928457</infon>
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<infon key="updated_at">2023-09-18T10:25:01Z</infon>
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<location offset="306" length="4"/>
<text>Xylp</text>
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<infon key="score">0.99911076</infon>
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<infon key="updated_at">2023-09-18T10:25:08Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>arabinofuranose</text>
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<infon key="updated_at">2023-09-18T10:25:15Z</infon>
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<text>Araf</text>
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<text>penta-modular enzyme</text>
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<infon key="updated_at">2023-09-18T10:25:29Z</infon>
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<location offset="414" length="7"/>
<text>CtXyl5A</text>
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<infon key="updated_at">2023-09-18T10:25:42Z</infon>
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<text>arabinoxylans</text>
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<infon key="updated_at">2023-09-18T10:25:48Z</infon>
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<text>crystal structure</text>
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<infon key="updated_at">2023-09-18T10:25:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="616" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="15">
<infon key="score">0.9980863</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="647" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="16">
<infon key="score">0.50939804</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="663" length="7"/>
<text>ligands</text>
</annotation>
<annotation id="17">
<infon key="score">0.9991667</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="767" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="18">
<infon key="score">0.99892145</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="800" length="41"/>
<text>non-catalytic carbohydrate binding module</text>
</annotation>
<annotation id="19">
<infon key="score">0.99877536</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="871" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="20">
<infon key="score">0.99855906</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="929" length="9"/>
<text>structure</text>
</annotation>
<annotation id="21">
<infon key="score">0.9979739</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="953" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="22">
<infon key="score">0.998847</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="969" length="50"/>
<text>Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp</text>
</annotation>
<annotation id="23">
<infon key="score">0.9983198</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1036" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="24">
<infon key="score">0.9865536</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1069" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="25">
<infon key="score">0.9988514</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1083" length="11"/>
<text>active site</text>
</annotation>
<annotation id="26">
<infon key="score">0.9991247</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:55:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1113" length="6"/>
<text>pocket</text>
</annotation>
<annotation id="27">
<infon key="score">0.9987526</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:55:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1121" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="28">
<infon key="score">0.9985621</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1154" length="16"/>
<text>catalytic center</text>
</annotation>
<annotation id="29">
<infon key="score">0.99883324</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:55:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1176" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="30">
<infon key="score">0.9991928</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1205" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="31">
<infon key="score">0.9991721</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1214" length="4"/>
<text>Arap</text>
</annotation>
<annotation id="32">
<infon key="score">0.9986651</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1258" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="33">
<infon key="score">0.9984883</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1269" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="34">
<infon key="score">0.99220085</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1308" length="20"/>
<text>Alanine substitution</text>
</annotation>
<annotation id="35">
<infon key="score">0.99950814</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1332" length="5"/>
<text>Glu68</text>
</annotation>
<annotation id="36">
<infon key="score">0.99953973</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1339" length="5"/>
<text>Tyr92</text>
</annotation>
<annotation id="37">
<infon key="score">0.99952006</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1349" length="6"/>
<text>Asn139</text>
</annotation>
<annotation id="38">
<infon key="score">0.9983119</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1377" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="39">
<infon key="score">0.9982514</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1391" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="40">
<infon key="score">0.99881476</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:55:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1417" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="41">
<infon key="score">0.9989426</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1474" length="11"/>
<text>active site</text>
</annotation>
<annotation id="42">
<infon key="score">0.9970607</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1491" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="43">
<infon key="score">0.99551225</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:48:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1575" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="44">
<infon key="score">0.9989027</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1610" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="45">
<infon key="score">0.9959501</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="1644" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="46">
<infon key="score">0.9521629</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1719" length="13"/>
<text>endo-xylanase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">title_1</infon>
<offset>1757</offset>
<text>Introduction</text>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1770</offset>
<text>The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.</text>
<annotation id="47">
<infon key="score">0.9988167</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1774" length="5"/>
<text>plant</text>
</annotation>
<annotation id="48">
<infon key="score">0.9986657</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:43:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1881" length="14"/>
<text>microorganisms</text>
</annotation>
<annotation id="49">
<infon key="score">0.9988594</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2270" length="5"/>
<text>plant</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2425</offset>
<text>An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.</text>
<annotation id="50">
<infon key="score">0.9986474</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2470" length="5"/>
<text>plant</text>
</annotation>
<annotation id="51">
<infon key="score">0.97633845</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2501" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="52">
<infon key="score">0.90193385</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2558" length="14"/>
<text>polysaccharide</text>
</annotation>
<annotation id="53">
<infon key="score">0.99882466</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:55Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2597" length="14"/>
<text>β-1,4-d-xylose</text>
</annotation>
<annotation id="1005">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:15Z</infon>
<location offset="2630" length="8"/>
<text>pyranose</text>
</annotation>
<annotation id="54">
<infon key="score">0.99762434</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2654" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="55">
<infon key="score">0.99699223</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2690" length="28"/>
<text>4-O-methyl-d-glucuronic acid</text>
</annotation>
<annotation id="56">
<infon key="score">0.99859554</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2720" length="4"/>
<text>GlcA</text>
</annotation>
<annotation id="57">
<infon key="score">0.9988452</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2751" length="19"/>
<text>α-l-arabinofuranose</text>
</annotation>
<annotation id="58">
<infon key="score">0.99826837</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2772" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="59">
<infon key="score">0.92519265</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2800" length="14"/>
<text>polysaccharide</text>
</annotation>
<annotation id="60">
<infon key="score">0.99387217</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2868" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="61">
<infon key="score">0.99838805</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2922" length="12"/>
<text>ferulic acid</text>
</annotation>
<annotation id="62">
<infon key="score">0.72233874</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="2990" length="13"/>
<text>hemicellulose</text>
</annotation>
<annotation id="899">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:37Z</infon>
<location offset="3008" length="6"/>
<text>lignin</text>
</annotation>
<annotation id="63">
<infon key="score">0.48770082</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3041" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="64">
<infon key="score">0.99851996</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3063" length="5"/>
<text>plant</text>
</annotation>
<annotation id="65">
<infon key="score">0.99830014</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3165" length="5"/>
<text>plant</text>
</annotation>
<annotation id="66">
<infon key="score">0.9969138</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:43:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3207" length="6"/>
<text>cereal</text>
</annotation>
<annotation id="67">
<infon key="score">0.70049024</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3222" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="68">
<infon key="score">0.99178773</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3298" length="6"/>
<text>sugars</text>
</annotation>
<annotation id="69">
<infon key="score">0.998679</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3315" length="18"/>
<text>l- and d-galactose</text>
</annotation>
<annotation id="70">
<infon key="score">0.9987858</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3338" length="13"/>
<text>β- and α-Xylp</text>
</annotation>
<annotation id="71">
<infon key="score">0.9947548</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:43:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3376" length="6"/>
<text>cereal</text>
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<annotation id="72">
<infon key="score">0.57896805</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3390" length="6"/>
<text>xylans</text>
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<annotation id="73">
<infon key="score">0.99786454</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3420" length="4"/>
<text>Xylp</text>
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<annotation id="74">
<infon key="score">0.93305</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3495" length="6"/>
<text>sugars</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3517</offset>
<text>Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.</text>
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<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3572" length="5"/>
<text>plant</text>
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<annotation id="76">
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<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:43:53Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>microorganisms</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3670" length="32"/>
<text>polysaccharide-degrading enzymes</text>
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<annotation id="78">
<infon key="score">0.9984101</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3714" length="20"/>
<text>glycoside hydrolases</text>
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<annotation id="79">
<infon key="score">0.9986268</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3745" length="21"/>
<text>polysaccharide lyases</text>
</annotation>
<annotation id="80">
<infon key="score">0.99855816</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3768" length="22"/>
<text>carbohydrate esterases</text>
</annotation>
<annotation id="81">
<infon key="score">0.99858</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3796" length="35"/>
<text>lytic polysaccharide monooxygenases</text>
</annotation>
<annotation id="82">
<infon key="score">0.99558616</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3839" length="27"/>
<text>carbohydrate active enzymes</text>
</annotation>
<annotation id="83">
<infon key="score">0.99593437</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3946" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="84">
<infon key="score">0.99739957</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3988" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="85">
<infon key="score">0.99709374</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4012" length="21"/>
<text>endo-acting xylanases</text>
</annotation>
<annotation id="86">
<infon key="score">0.9923669</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4072" length="19"/>
<text>glycoside hydrolase</text>
</annotation>
<annotation id="87">
<infon key="score">0.99262285</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4093" length="2"/>
<text>GH</text>
</annotation>
<annotation id="88">
<infon key="score">0.9738185</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4096" length="1"/>
<text>5</text>
</annotation>
<annotation id="89">
<infon key="score">0.9936046</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4107" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="90">
<infon key="score">0.99484766</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4116" length="4"/>
<text>GH11</text>
</annotation>
<annotation id="91">
<infon key="score">0.9940958</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4156" length="3"/>
<text>GH8</text>
</annotation>
<annotation id="92">
<infon key="score">0.9956683</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4193" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="93">
<infon key="score">0.99571615</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4272" length="14"/>
<text>polysaccharide</text>
</annotation>
<annotation id="94">
<infon key="score">0.99883527</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:32:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4337" length="16"/>
<text>α-glucuronidases</text>
</annotation>
<annotation id="95">
<infon key="score">0.9988317</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:32:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4355" length="22"/>
<text>α-arabinofuranosidases</text>
</annotation>
<annotation id="96">
<infon key="score">0.9990785</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:32:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4383" length="9"/>
<text>esterases</text>
</annotation>
<annotation id="97">
<infon key="score">0.99923325</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4398" length="9"/>
<text>xylanases</text>
</annotation>
<annotation id="98">
<infon key="score">0.9843167</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4519" length="13"/>
<text>hemicellulose</text>
</annotation>
<annotation id="99">
<infon key="score">0.99851674</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4538" length="4"/>
<text>GH30</text>
</annotation>
<annotation id="100">
<infon key="score">0.99904984</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4543" length="18"/>
<text>glucuronoxylanases</text>
</annotation>
<annotation id="101">
<infon key="score">0.99920505</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4574" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="102">
<infon key="score">0.9013893</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4579" length="8"/>
<text>bound at</text>
</annotation>
<annotation id="103">
<infon key="score">0.99698335</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:55:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4592" length="2"/>
<text>−2</text>
</annotation>
<annotation id="104">
<infon key="score">0.94409853</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4608" length="4"/>
<text>GlcA</text>
</annotation>
<annotation id="105">
<infon key="score">0.9941329</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4655" length="20"/>
<text>glycoside hydrolases</text>
</annotation>
<annotation id="1015">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:56:02Z</infon>
<location offset="4687" length="18"/>
<text>subsites −1 and +1</text>
</annotation>
<annotation id="106">
<infon key="score">0.9901659</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:56:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4711" length="8"/>
<text>subsites</text>
</annotation>
<annotation id="107">
<infon key="score">0.9971566</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4865" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="108">
<infon key="score">0.9978956</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4869" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="109">
<infon key="score">0.99816823</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4886" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="110">
<infon key="score">0.9986105</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4908" length="24"/>
<text>Clostridium thermocellum</text>
</annotation>
<annotation id="111">
<infon key="score">0.99725866</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4970" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="112">
<infon key="score">0.99785656</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4990" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="113">
<infon key="score">0.99804723</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5063" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="114">
<infon key="score">0.999025</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5090" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="115">
<infon key="score">0.9891261</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5095" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="116">
<infon key="score">0.99886334</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5108" length="11"/>
<text>active site</text>
</annotation>
<annotation id="117">
<infon key="score">0.9914019</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:56:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5121" length="10"/>
<text>−1 subsite</text>
</annotation>
<annotation id="118">
<infon key="score">0.99909043</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5171" length="13"/>
<text>arabinoxylans</text>
</annotation>
<annotation id="119">
<infon key="score">0.99802303</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5208" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="120">
<infon key="score">0.99847084</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:56:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5236" length="8"/>
<text>subsites</text>
</annotation>
<annotation id="121">
<infon key="score">0.99856865</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5259" length="11"/>
<text>active site</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>5280</offset>
<text>CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.</text>
<annotation id="122">
<infon key="score">0.99918216</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5280" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="123">
<infon key="score">0.67755586</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5344" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="124">
<infon key="score">0.9980315</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5348" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="125">
<infon key="score">0.860335</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5366" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="126">
<infon key="score">0.9982284</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5380" length="42"/>
<text>non-catalytic carbohydrate binding modules</text>
</annotation>
<annotation id="127">
<infon key="score">0.9514713</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5424" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="128">
<infon key="score">0.3509473</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5452" length="1"/>
<text>6</text>
</annotation>
<annotation id="129">
<infon key="score">0.6156626</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5455" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="130">
<infon key="score">0.29874778</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5464" length="2"/>
<text>13</text>
</annotation>
<annotation id="131">
<infon key="score">0.874645</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5468" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="132">
<infon key="score">0.6269602</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5482" length="2"/>
<text>62</text>
</annotation>
<annotation id="133">
<infon key="score">0.8562604</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5486" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="134">
<infon key="score">0.9989775</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5496" length="18"/>
<text>fibronectin type 3</text>
</annotation>
<annotation id="135">
<infon key="score">0.9988217</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5516" length="3"/>
<text>Fn3</text>
</annotation>
<annotation id="136">
<infon key="score">0.99874604</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5546" length="8"/>
<text>dockerin</text>
</annotation>
<annotation id="137">
<infon key="score">0.999186</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5590" length="3"/>
<text>Fn3</text>
</annotation>
<annotation id="138">
<infon key="score">0.9520046</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5645" length="22"/>
<text>ligand-binding modules</text>
</annotation>
<annotation id="139">
<infon key="score">0.9833201</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5743" length="28"/>
<text>cellulose-disrupting modules</text>
</annotation>
<annotation id="140">
<infon key="score">0.9987042</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5841" length="8"/>
<text>dockerin</text>
</annotation>
<annotation id="141">
<infon key="score">0.99853694</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:32Z</infon>
<infon key="identifier">GO:</infon>
<location offset="5886" length="11"/>
<text>cellulosome</text>
</annotation>
<annotation id="142">
<infon key="score">0.98729646</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5913" length="5"/>
<text>plant</text>
</annotation>
<annotation id="143">
<infon key="score">0.99846244</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5975" length="15"/>
<text>C. thermocellum</text>
</annotation>
<annotation id="144">
<infon key="score">0.57434464</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5992" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="145">
<infon key="score">0.6251633</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6010" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="146">
<infon key="score">0.59856856</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6021" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="147">
<infon key="score">0.99911016</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6038" length="17"/>
<text>d-galactopyranose</text>
</annotation>
<annotation id="148">
<infon key="score">0.99909335</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6060" length="17"/>
<text>l-arabinopyranose</text>
</annotation>
<annotation id="149">
<infon key="score">0.98460996</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6099" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="905">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:04Z</infon>
<location offset="6111" length="3"/>
<text>Fn3</text>
</annotation>
<annotation id="150">
<infon key="score">0.9985752</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6272" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="151">
<infon key="score">0.99908304</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:39:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6293" length="6"/>
<text>mature</text>
</annotation>
<annotation id="152">
<infon key="score">0.9991793</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="6300" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="153">
<infon key="score">0.99882346</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:39:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6308" length="7"/>
<text>lacking</text>
</annotation>
<annotation id="907">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:10Z</infon>
<location offset="6331" length="8"/>
<text>dockerin</text>
</annotation>
<annotation id="154">
<infon key="score">0.7470989</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:39:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6348" length="11"/>
<text>CtXyl5A-Doc</text>
</annotation>
<annotation id="893">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<location offset="6377" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="910">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:19Z</infon>
<location offset="6393" length="7"/>
<text>ligands</text>
</annotation>
<annotation id="155">
<infon key="score">0.9989073</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6559" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="156">
<infon key="score">0.99873036</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6603" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="157">
<infon key="score">0.99821556</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6612" length="4"/>
<text>GH11</text>
</annotation>
<annotation id="158">
<infon key="score">0.9986395</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6617" length="14"/>
<text>endo-xylanases</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440001.jpg</infon>
<infon key="id">F1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>6633</offset>
<text>Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains.</text>
<annotation id="958">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="6659" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="159">
<infon key="score">0.7534902</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:41:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6695" length="2"/>
<text>GH</text>
</annotation>
<annotation id="160">
<infon key="score">0.9863507</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6699" length="3"/>
<text>CBM</text>
</annotation>
<annotation id="161">
<infon key="score">0.99045384</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6707" length="2"/>
<text>CE</text>
</annotation>
<annotation id="162">
<infon key="score">0.99766254</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6739" length="19"/>
<text>glycoside hydrolase</text>
</annotation>
<annotation id="163">
<infon key="score">0.7450091</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:42:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6760" length="27"/>
<text>carbohydrate binding module</text>
</annotation>
<annotation id="164">
<infon key="score">0.9983574</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:42:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6792" length="21"/>
<text>carbohydrate esterase</text>
</annotation>
<annotation id="911">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:41:42Z</infon>
<location offset="6838" length="11"/>
<text>Laminin_3_G</text>
</annotation>
<annotation id="165">
<infon key="score">0.99794406</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:41:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6872" length="33"/>
<text>concanavalin A lectin superfamily</text>
</annotation>
<annotation id="166">
<infon key="score">0.998723</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6911" length="3"/>
<text>FN3</text>
</annotation>
<annotation id="167">
<infon key="score">0.9979355</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="6925" length="25"/>
<text>fibronectin type 3 domain</text>
</annotation>
<annotation id="908">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:10Z</infon>
<location offset="6975" length="8"/>
<text>dockerin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>6993</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_4</infon>
<offset>7001</offset>
<text>Substrate Specificity of CtXyl5A</text>
<annotation id="168">
<infon key="score">0.99469876</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7026" length="7"/>
<text>CtXyl5A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>7034</offset>
<text>Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.</text>
<annotation id="169">
<infon key="score">0.999027</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="7063" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="170">
<infon key="score">0.9967934</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:34:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7077" length="30"/>
<text>arabinoxylan-specific xylanase</text>
</annotation>
<annotation id="171">
<infon key="score">0.9990753</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7123" length="20"/>
<text>xylooligosaccharides</text>
</annotation>
<annotation id="172">
<infon key="score">0.99847776</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7152" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="173">
<infon key="score">0.99852186</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7192" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="174">
<infon key="score">0.8807422</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:44:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7234" length="5"/>
<text>wheat</text>
</annotation>
<annotation id="175">
<infon key="score">0.99000096</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:44:41Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7248" length="13"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:23Z</infon>
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<infon key="updated_at">2023-09-18T10:45:27Z</infon>
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<location offset="7286" length="3"/>
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<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
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<location offset="7438" length="10"/>
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<location offset="7514" length="16"/>
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<location offset="7687" length="10"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-18T10:24:32Z</infon>
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<location offset="7837" length="6"/>
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<infon key="updated_at">2023-09-18T10:43:47Z</infon>
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<location offset="7849" length="6"/>
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<infon key="updated_at">2023-09-18T10:25:15Z</infon>
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<location offset="8035" length="4"/>
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<location offset="8044" length="4"/>
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<location offset="8069" length="5"/>
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<infon key="updated_at">2023-09-18T10:46:27Z</infon>
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<location offset="8076" length="5"/>
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<infon key="updated_at">2023-09-18T10:46:32Z</infon>
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<location offset="8087" length="5"/>
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<infon key="updated_at">2023-09-18T10:26:33Z</infon>
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<infon key="updated_at">2023-09-18T10:45:08Z</infon>
<location offset="8242" length="4"/>
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<location offset="8252" length="5"/>
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<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="2" colspan="1">Enzyme</th><th align="center" rowspan="2" colspan="1">Variant</th><th align="center" rowspan="1" colspan="3"><italic>k</italic><sub>cat</sub>/<italic>K<sub>m</sub></italic><hr/></th></tr><tr><th align="center" rowspan="1" colspan="1">WAX</th><th align="center" rowspan="1" colspan="1">RAX</th><th align="center" rowspan="1" colspan="1">CX</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="3"><italic>min</italic><sup>−<italic>1</italic></sup>
<italic>mg</italic><sup>−<italic>1</italic></sup>
<italic>ml</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3-CBM62</td><td align="left" rowspan="1" colspan="1">800</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">460</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3</td><td align="left" rowspan="1" colspan="1">1,232</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">659</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13</td><td align="left" rowspan="1" colspan="1">1,307</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">620</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6</td><td align="left" rowspan="1" colspan="1">488</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">102</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: E68A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: Y92A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N135A</td><td align="left" rowspan="1" colspan="1">260</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N139A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ac</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">628</td><td align="left" rowspan="1" colspan="1">1,641</td><td align="left" rowspan="1" colspan="1">289</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Gp</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">2,600</td><td align="left" rowspan="1" colspan="1">9,986</td><td align="left" rowspan="1" colspan="1">314</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">D45A</td><td align="left" rowspan="1" colspan="1">102</td><td align="left" rowspan="1" colspan="1">203</td><td align="left" rowspan="1" colspan="1">23</td></tr></tbody></table>
</infon>
<offset>8846</offset>
<text>Enzyme Variant kcat/Km WAX RAX CX min−1mg−1ml CtXyl5A CtGH5-CBM6-CBM13-Fn3-CBM62 800 ND 460 CtXyl5A CtGH5-CBM6-CBM13-Fn3 1,232 ND 659 CtXyl5A CtGH5-CBM6-CBM13 1,307 ND 620 CtXyl5A CtGH5-CBM6 488 ND 102 CtXyl5A CtGH5-CBM6: E68A NA NA NA CtXyl5A CtGH5-CBM6: Y92A NA NA NA CtXyl5A CtGH5-CBM6: N135A 260 ND ND CtXyl5A CtGH5-CBM6: N139A NA NA NA AcGH5 Wild type 628 1,641 289 GpGH5 Wild type 2,600 9,986 314 VbGH5 Wild type ND ND ND VbGH5 D45A 102 203 23 </text>
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<infon key="updated_at">2023-09-18T10:49:27Z</infon>
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<passage>
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<infon key="type">paragraph</infon>
<offset>9333</offset>
<text>To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.</text>
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<infon key="updated_at">2023-09-18T13:35:57Z</infon>
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<infon key="updated_at">2023-09-18T13:57:04Z</infon>
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<infon key="updated_at">2023-09-18T10:58:49Z</infon>
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<text>oligosaccharide</text>
</annotation>
<annotation id="999">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:32Z</infon>
<location offset="9809" length="15"/>
<text>oligosaccharide</text>
</annotation>
<annotation id="254">
<infon key="score">0.9654604</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:58:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="9838" length="44"/>
<text>Positive mode electrospray mass spectrometry</text>
</annotation>
<annotation id="949">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:16:00Z</infon>
<location offset="9990" length="7"/>
<text>pentose</text>
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<annotation id="950">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:16:00Z</infon>
<location offset="9998" length="7"/>
<text>pentose</text>
</annotation>
<annotation id="255">
<infon key="score">0.7560745</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10006" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="256">
<infon key="score">0.8210523</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10092" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="257">
<infon key="score">0.9970212</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:59:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10220" length="14"/>
<text>TFA hydrolysis</text>
</annotation>
<annotation id="258">
<infon key="score">0.9957487</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10245" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="259">
<infon key="score">0.9935015</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10256" length="9"/>
<text>arabinose</text>
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<annotation id="260">
<infon key="score">0.9950472</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10309" length="16"/>
<text>oligosaccharides</text>
</annotation>
<annotation id="261">
<infon key="score">0.9897843</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:04:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10341" length="13"/>
<text>disaccharides</text>
</annotation>
<annotation id="262">
<infon key="score">0.9872656</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10378" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="263">
<infon key="score">0.97808754</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10425" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="264">
<infon key="score">0.9635059</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10447" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="947">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:15:03Z</infon>
<location offset="10484" length="31"/>
<text>nonspecific arabinofuranosidase</text>
</annotation>
<annotation id="265">
<infon key="score">0.9518543</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:54:51Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10517" length="8"/>
<text>CjAbf51A</text>
</annotation>
<annotation id="997">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:31Z</infon>
<location offset="10551" length="15"/>
<text>oligosaccharide</text>
</annotation>
<annotation id="266">
<infon key="score">0.9971559</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10596" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="267">
<infon key="score">0.9963881</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10607" length="9"/>
<text>arabinose</text>
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<annotation id="944">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<location offset="10634" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="268">
<infon key="score">0.9972711</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10650" length="6"/>
<text>xylose</text>
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<annotation id="269">
<infon key="score">0.9962746</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10661" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="270">
<infon key="score">0.9963164</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:55:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10700" length="16"/>
<text>β-1,3-xylosidase</text>
</annotation>
<annotation id="271">
<infon key="score">0.5966584</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:55:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10718" length="4"/>
<text>XynB</text>
</annotation>
<annotation id="1000">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:32Z</infon>
<location offset="10734" length="15"/>
<text>oligosaccharide</text>
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<annotation id="272">
<infon key="score">0.9976882</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10757" length="6"/>
<text>xylose</text>
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<annotation id="945">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<location offset="10805" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="273">
<infon key="score">0.9992453</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:06Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10818" length="15"/>
<text>β-1,3-xylobiose</text>
</annotation>
<annotation id="274">
<infon key="score">0.97893924</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:59:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10880" length="25"/>
<text>β-1,4-specific xylosidase</text>
</annotation>
<annotation id="1001">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:32Z</infon>
<location offset="10919" length="15"/>
<text>oligosaccharide</text>
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<annotation id="1002">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:32Z</infon>
<location offset="10940" length="15"/>
<text>oligosaccharide</text>
</annotation>
<annotation id="275">
<infon key="score">0.9987957</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11019" length="10"/>
<text>−2* pocket</text>
</annotation>
<annotation id="276">
<infon key="score">0.9992246</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:55:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11121" length="26"/>
<text>β-1,4-xylooligosaccharides</text>
</annotation>
<annotation id="277">
<infon key="score">0.999252</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:55:27Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11167" length="9"/>
<text>Araf-Xylp</text>
</annotation>
<annotation id="278">
<infon key="score">0.9992756</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11181" length="13"/>
<text>Xyl-β-1,3-Xyl</text>
</annotation>
<annotation id="279">
<infon key="score">0.9986477</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11251" length="10"/>
<text>−2 subsite</text>
</annotation>
<annotation id="280">
<infon key="score">0.9981468</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11331" length="21"/>
<text>endo-acting xylanases</text>
</annotation>
<annotation id="281">
<infon key="score">0.99076325</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11364" length="7"/>
<text>subsite</text>
</annotation>
<annotation id="282">
<infon key="score">0.99634886</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11448" length="3"/>
<text>−2*</text>
</annotation>
<annotation id="283">
<infon key="score">0.9985508</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11521" length="10"/>
<text>−2 subsite</text>
</annotation>
<annotation id="951">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:18:33Z</infon>
<location offset="11590" length="32"/>
<text>(Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl</text>
</annotation>
<annotation id="284">
<infon key="score">0.9294588</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11623" length="16"/>
<text>oligosaccharides</text>
</annotation>
<annotation id="888">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<location offset="11645" length="13"/>
<text>arabinoxylans</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440002.jpg</infon>
<infon key="id">F2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>11762</offset>
<text>Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.</text>
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<infon key="score">0.99689424</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11784" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="286">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11830" length="2"/>
<text>CX</text>
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<annotation id="287">
<infon key="score">0.99636436</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:59:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11882" length="29"/>
<text>size exclusion chromatography</text>
</annotation>
<annotation id="288">
<infon key="score">0.9975695</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:58:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11928" length="5"/>
<text>HPAEC</text>
</annotation>
<annotation id="289">
<infon key="score">0.9924101</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:14:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11956" length="6"/>
<text>ESI-MS</text>
</annotation>
<annotation id="946">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:15:02Z</infon>
<location offset="12014" length="31"/>
<text>nonspecific arabinofuranosidase</text>
</annotation>
<annotation id="290">
<infon key="score">0.9880223</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:54:52Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12047" length="8"/>
<text>CjAbf51A</text>
</annotation>
<annotation id="948">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:15:31Z</infon>
<location offset="12063" length="14"/>
<text>GH3 xylosidase</text>
</annotation>
<annotation id="291">
<infon key="score">0.99361575</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:55:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12079" length="4"/>
<text>XynB</text>
</annotation>
<annotation id="292">
<infon key="score">0.99745184</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12125" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="293">
<infon key="score">0.99641883</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12136" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="294">
<infon key="score">0.9981957</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:16:00Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12179" length="7"/>
<text>pentose</text>
</annotation>
<annotation id="295">
<infon key="score">0.76520646</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12187" length="12"/>
<text>disaccharide</text>
</annotation>
<annotation id="296">
<infon key="score">0.58194405</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:14:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12277" length="6"/>
<text>ESI-MS</text>
</annotation>
<annotation id="297">
<infon key="score">0.9983165</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:15:59Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12297" length="7"/>
<text>pentose</text>
</annotation>
<annotation id="298">
<infon key="score">0.96897036</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12305" length="12"/>
<text>disaccharide</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_4</infon>
<offset>12357</offset>
<text>Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands</text>
<annotation id="299">
<infon key="score">0.9987371</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12357" length="17"/>
<text>Crystal Structure</text>
</annotation>
<annotation id="300">
<infon key="score">0.998405</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12382" length="16"/>
<text>Catalytic Module</text>
</annotation>
<annotation id="301">
<infon key="score">0.95691925</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12402" length="7"/>
<text>CtXyl5A</text>
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<annotation id="302">
<infon key="score">0.99861926</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12410" length="15"/>
<text>in Complex with</text>
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<annotation id="303">
<infon key="score">0.46196067</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12426" length="7"/>
<text>Ligands</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12434</offset>
<text>To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12503" length="7"/>
<text>CtXyl5A</text>
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<annotation id="305">
<infon key="score">0.9986395</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12516" length="17"/>
<text>crystal structure</text>
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<annotation id="306">
<infon key="score">0.9989794</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:17:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12577" length="23"/>
<text>substrate binding cleft</text>
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<infon key="score">0.99862534</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12618" length="11"/>
<text>−2* subsite</text>
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<annotation id="308">
<infon key="score">0.99843544</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:50:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12682" length="9"/>
<text>structure</text>
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<annotation id="309">
<infon key="score">0.7080453</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12699" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="1042">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:29:50Z</infon>
<location offset="12718" length="12"/>
<text>CtGH5-CtCBM6</text>
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<annotation id="310">
<infon key="score">0.9989565</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12731" length="15"/>
<text>in complex with</text>
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<annotation id="311">
<infon key="score">0.99890447</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12747" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="312">
<infon key="score">0.99898124</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12757" length="8"/>
<text>bound in</text>
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<annotation id="313">
<infon key="score">0.99854136</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12770" length="10"/>
<text>−2* pocket</text>
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<annotation id="314">
<infon key="score">0.9988469</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:17:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12801" length="5"/>
<text>bound</text>
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<annotation id="315">
<infon key="score">0.9989888</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12807" length="9"/>
<text>arabinose</text>
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<annotation id="1004">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:14Z</infon>
<location offset="12828" length="8"/>
<text>pyranose</text>
</annotation>
<annotation id="1008">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:26Z</infon>
<location offset="12869" length="8"/>
<text>furanose</text>
</annotation>
<annotation id="316">
<infon key="score">0.9973653</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12892" length="13"/>
<text>arabinoxylans</text>
</annotation>
<annotation id="317">
<infon key="score">0.99867976</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12932" length="11"/>
<text>active site</text>
</annotation>
<annotation id="318">
<infon key="score">0.998457</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12944" length="10"/>
<text>−1 subsite</text>
</annotation>
<annotation id="319">
<infon key="score">0.9984829</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:17:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12974" length="5"/>
<text>bound</text>
</annotation>
<annotation id="320">
<infon key="score">0.99893314</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12980" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="321">
<infon key="score">0.9965403</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13041" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="322">
<infon key="score">0.9991899</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13121" length="4"/>
<text>Arap</text>
</annotation>
<annotation id="323">
<infon key="score">0.99857426</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13152" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="324">
<infon key="score">0.99846435</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13185" length="6"/>
<text>pocket</text>
</annotation>
<annotation id="325">
<infon key="score">0.99902916</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13222" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="326">
<infon key="score">0.9983833</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:21:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13247" length="7"/>
<text>soaking</text>
</annotation>
<annotation id="327">
<infon key="score">0.99923503</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:21:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13255" length="3"/>
<text>apo</text>
</annotation>
<annotation id="328">
<infon key="score">0.48547208</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:50:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13259" length="8"/>
<text>crystals</text>
</annotation>
<annotation id="329">
<infon key="score">0.99908113</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13273" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="1003">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:56Z</infon>
<location offset="13296" length="7"/>
<text>pentose</text>
</annotation>
<annotation id="330">
<infon key="score">0.5487963</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13304" length="5"/>
<text>sugar</text>
</annotation>
<annotation id="331">
<infon key="score">0.9981328</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13315" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="332">
<infon key="score">0.9983646</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:57:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13328" length="11"/>
<text>−2* subsite</text>
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<annotation id="1006">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:15Z</infon>
<location offset="13347" length="8"/>
<text>pyranose</text>
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<annotation id="333">
<infon key="score">0.9988084</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:50:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13386" length="18"/>
<text>crystal structures</text>
</annotation>
<annotation id="334">
<infon key="score">0.9991225</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13484" length="15"/>
<text>β-1,3-xylobiose</text>
</annotation>
<annotation id="335">
<infon key="score">0.9822428</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13505" length="2"/>
<text>CX</text>
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<annotation id="336">
<infon key="score">0.9982703</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:20Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13533" length="12"/>
<text>disaccharide</text>
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<annotation id="1017">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:09Z</infon>
<location offset="13561" length="19"/>
<text>−1 and −2* subsites</text>
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<annotation id="337">
<infon key="score">0.99872786</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:05:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13623" length="16"/>
<text>co-crystallizing</text>
</annotation>
<annotation id="338">
<infon key="score">0.99801016</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13644" length="20"/>
<text>nucleophile inactive</text>
</annotation>
<annotation id="339">
<infon key="score">0.99902153</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13665" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="340">
<infon key="score">0.9535321</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:09:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13672" length="10"/>
<text>CtGH5E279S</text>
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<annotation id="903">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:40Z</infon>
<location offset="13683" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="341">
<infon key="score">0.98482054</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13697" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="342">
<infon key="score">0.9992003</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:39:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13709" length="15"/>
<text>oligosaccharide</text>
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<annotation id="343">
<infon key="score">0.9991468</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:37Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13756" length="15"/>
<text>pentasaccharide</text>
</annotation>
<annotation id="344">
<infon key="score">0.9987526</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13772" length="8"/>
<text>bound to</text>
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<annotation id="345">
<infon key="score">0.99912447</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13804" length="18"/>
<text>β-1,4-xylotetraose</text>
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<annotation id="346">
<infon key="score">0.99879885</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13831" length="4"/>
<text>Araf</text>
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<annotation id="347">
<infon key="score">0.9987872</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13869" length="6"/>
<text>xylose</text>
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<annotation id="348">
<infon key="score">0.99923956</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13881" length="12"/>
<text>xylotetraose</text>
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<annotation id="349">
<infon key="score">0.9051479</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13912" length="17"/>
<text>subsites −1 to −4</text>
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<annotation id="350">
<infon key="score">0.9991596</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13938" length="4"/>
<text>Araf</text>
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<annotation id="351">
<infon key="score">0.9983297</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13950" length="10"/>
<text>−2* pocket</text>
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<annotation id="352">
<infon key="score">0.9958412</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:50:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13984" length="10"/>
<text>structures</text>
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<infon key="score">0.99901795</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14081" length="4"/>
<text>Arap</text>
</annotation>
<annotation id="354">
<infon key="score">0.9989477</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14087" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="355">
<infon key="score">0.9988612</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14097" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="356">
<infon key="score">0.9988979</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14110" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="357">
<infon key="score">0.99790555</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14123" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="358">
<infon key="score">0.9976653</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14181" length="6"/>
<text>pocket</text>
</annotation>
<annotation id="359">
<infon key="score">0.9838765</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14200" length="13"/>
<text>polar contact</text>
</annotation>
<annotation id="360">
<infon key="score">0.9994537</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14226" length="6"/>
<text>Asn139</text>
</annotation>
<annotation id="361">
<infon key="score">0.99667907</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14247" length="16"/>
<text>hydrogen bonding</text>
</annotation>
<annotation id="362">
<infon key="score">0.999443</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14285" length="6"/>
<text>Asn139</text>
</annotation>
<annotation id="363">
<infon key="score">0.99943477</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14314" length="6"/>
<text>Asn135</text>
</annotation>
<annotation id="364">
<infon key="score">0.99941826</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14353" length="6"/>
<text>Gly136</text>
</annotation>
<annotation id="365">
<infon key="score">0.99930656</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14371" length="5"/>
<text>Glu68</text>
</annotation>
<annotation id="366">
<infon key="score">0.99909437</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:40:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14393" length="4"/>
<text>Arap</text>
</annotation>
<annotation id="367">
<infon key="score">0.9990483</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14463" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="368">
<infon key="score">0.9990252</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14472" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="369">
<infon key="score">0.9971278</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14497" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="370">
<infon key="score">0.99928504</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14524" length="5"/>
<text>Glu68</text>
</annotation>
<annotation id="371">
<infon key="score">0.9993992</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14539" length="5"/>
<text>Tyr92</text>
</annotation>
<annotation id="372">
<infon key="score">0.97813207</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14558" length="21"/>
<text>parallel interactions</text>
</annotation>
<annotation id="1007">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:15Z</infon>
<location offset="14589" length="8"/>
<text>pyranose</text>
</annotation>
<annotation id="1009">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:27Z</infon>
<location offset="14601" length="8"/>
<text>furanose</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440003.jpg</infon>
<infon key="id">F3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>14671</offset>
<text>Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated.</text>
<annotation id="373">
<infon key="score">0.99794537</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14745" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="374">
<infon key="score">0.99496317</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14879" length="18"/>
<text>catalytic residues</text>
</annotation>
<annotation id="1018">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:46Z</infon>
<location offset="14906" length="7"/>
<text>mutated</text>
</annotation>
<annotation id="375">
<infon key="score">0.9964224</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:21:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14914" length="9"/>
<text>glutamate</text>
</annotation>
<annotation id="376">
<infon key="score">0.9966878</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:21:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14932" length="6"/>
<text>serine</text>
</annotation>
<annotation id="1043">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:30:17Z</infon>
<location offset="14959" length="10"/>
<text>CtGH5-CBM6</text>
</annotation>
<annotation id="377">
<infon key="score">0.96348566</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14970" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="378">
<infon key="score">0.9985078</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:51Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="14989" length="15"/>
<text>arabinopyranose</text>
</annotation>
<annotation id="1044">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:30:42Z</infon>
<location offset="15009" length="10"/>
<text>CtGH5-CBM6</text>
</annotation>
<annotation id="379">
<infon key="score">0.9178238</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15020" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="380">
<infon key="score">0.9992086</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15038" length="12"/>
<text>xylopyranose</text>
</annotation>
<annotation id="381">
<infon key="score">0.99710983</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:09:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15055" length="10"/>
<text>CtGH5E279S</text>
</annotation>
<annotation id="939">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:51:34Z</infon>
<location offset="15066" length="4"/>
<text>CBM6</text>
</annotation>
<annotation id="382">
<infon key="score">0.9848954</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15071" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="383">
<infon key="score">0.99923825</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:01Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15089" length="15"/>
<text>pentasaccharide</text>
</annotation>
<annotation id="384">
<infon key="score">0.99916303</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15106" length="17"/>
<text>β1,4-xylotetraose</text>
</annotation>
<annotation id="385">
<infon key="score">0.999108</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15132" length="6"/>
<text>l-Araf</text>
</annotation>
<annotation id="386">
<infon key="score">0.9986161</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15171" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="387">
<infon key="score">0.9975291</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15184" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="388">
<infon key="score">0.9933159</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:22:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15240" length="9"/>
<text>Densities</text>
</annotation>
<annotation id="952">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:22:32Z</infon>
<location offset="15275" length="48"/>
<text>maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15430</offset>
<text>The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.</text>
<annotation id="389">
<infon key="score">0.99893385</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15528" length="10"/>
<text>−2* pocket</text>
</annotation>
<annotation id="390">
<infon key="score">0.9966867</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15557" length="20"/>
<text>alanine substitution</text>
</annotation>
<annotation id="391">
<infon key="score">0.9985077</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:22:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15604" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="392">
<infon key="score">0.9990114</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15612" length="4"/>
<text>E68A</text>
</annotation>
<annotation id="393">
<infon key="score">0.9989949</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15618" length="4"/>
<text>Y92A</text>
</annotation>
<annotation id="394">
<infon key="score">0.99898463</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:54:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15628" length="5"/>
<text>N139A</text>
</annotation>
<annotation id="395">
<infon key="score">0.99903464</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15643" length="8"/>
<text>inactive</text>
</annotation>
<annotation id="396">
<infon key="score">0.99899477</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15787" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="397">
<infon key="score">0.9990736</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15836" length="5"/>
<text>N135A</text>
</annotation>
<annotation id="398">
<infon key="score">0.99906486</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15851" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="399">
<infon key="score">0.9993063</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15932" length="6"/>
<text>Asn135</text>
</annotation>
<annotation id="400">
<infon key="score">0.9992543</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15993" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="401">
<infon key="score">0.99923706</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16001" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="402">
<infon key="score">0.9989507</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:58:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16013" length="10"/>
<text>−2* pocket</text>
</annotation>
<annotation id="926">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:48:32Z</infon>
<location offset="16032" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="403">
<infon key="score">0.99911714</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16053" length="11"/>
<text>active site</text>
</annotation>
<annotation id="404">
<infon key="score">0.9991146</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16072" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="405">
<infon key="score">0.99835086</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16105" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="406">
<infon key="score">0.9980102</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16143" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="407">
<infon key="score">0.9981377</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16153" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="1012">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:50:52Z</infon>
<location offset="16203" length="4"/>
<text>kcat</text>
</annotation>
<annotation id="1013">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:00Z</infon>
<location offset="16208" length="2"/>
<text>Km</text>
</annotation>
<annotation id="408">
<infon key="score">0.9909752</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16215" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="409">
<infon key="score">0.9985014</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16231" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="410">
<infon key="score">0.7723853</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16266" length="2"/>
<text>CX</text>
</annotation>
<annotation id="411">
<infon key="score">0.99858814</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16280" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="412">
<infon key="score">0.99835324</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16335" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="413">
<infon key="score">0.926579</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16366" length="2"/>
<text>CX</text>
</annotation>
<annotation id="414">
<infon key="score">0.99903345</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16418" length="15"/>
<text>arabinoxylanase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>16441</offset>
<text>In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.</text>
<annotation id="415">
<infon key="score">0.99896586</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16448" length="11"/>
<text>active site</text>
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<annotation id="416">
<infon key="score">0.99930596</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16463" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="417">
<infon key="score">0.9991194</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16475" length="8"/>
<text>α-d-Xylp</text>
</annotation>
<annotation id="418">
<infon key="score">0.99690115</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16594" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="419">
<infon key="score">0.9994248</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16625" length="6"/>
<text>His253</text>
</annotation>
<annotation id="420">
<infon key="score">0.9993506</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:24:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16643" length="6"/>
<text>Glu171</text>
</annotation>
<annotation id="421">
<infon key="score">0.9857984</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16698" length="13"/>
<text>polar contact</text>
</annotation>
<annotation id="422">
<infon key="score">0.99934465</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16727" length="6"/>
<text>Tyr255</text>
</annotation>
<annotation id="423">
<infon key="score">0.9993443</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16744" length="6"/>
<text>Ser279</text>
</annotation>
<annotation id="424">
<infon key="score">0.99672544</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16795" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="425">
<infon key="score">0.9993864</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:24:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16822" length="6"/>
<text>Asn170</text>
</annotation>
<annotation id="426">
<infon key="score">0.9994099</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16839" length="5"/>
<text>Tyr92</text>
</annotation>
<annotation id="427">
<infon key="score">0.999243</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16860" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="428">
<infon key="score">0.9981232</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16872" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="429">
<infon key="score">0.9942347</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16893" length="13"/>
<text>polar contact</text>
</annotation>
<annotation id="430">
<infon key="score">0.9994549</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16919" length="6"/>
<text>Asn139</text>
</annotation>
<annotation id="431">
<infon key="score">0.9965925</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:24:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16949" length="15"/>
<text>hydrogens bonds</text>
</annotation>
<annotation id="432">
<infon key="score">0.9993518</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:23:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16980" length="6"/>
<text>Tyr255</text>
</annotation>
<annotation id="433">
<infon key="score">0.9992919</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16992" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="434">
<infon key="score">0.99828696</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17004" length="11"/>
<text>active site</text>
</annotation>
<annotation id="435">
<infon key="score">0.8674073</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:24:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17029" length="28"/>
<text>parallel apolar interactions</text>
</annotation>
<annotation id="436">
<infon key="score">0.9994276</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:24:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17063" length="6"/>
<text>Phe310</text>
</annotation>
<annotation id="437">
<infon key="score">0.9986855</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17100" length="11"/>
<text>active site</text>
</annotation>
<annotation id="438">
<infon key="score">0.9959131</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17115" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="439">
<infon key="score">0.99930716</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17133" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="440">
<infon key="score">0.99881834</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17157" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="441">
<infon key="score">0.9991805</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:34:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17185" length="13"/>
<text>endoglucanase</text>
</annotation>
<annotation id="442">
<infon key="score">0.9994155</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17199" length="7"/>
<text>BaCel5A</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440004.jpg</infon>
<infon key="id">F4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>17270</offset>
<text>Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.
A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds.</text>
<annotation id="443">
<infon key="score">0.99856913</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17321" length="17"/>
<text>negative subsites</text>
</annotation>
<annotation id="444">
<infon key="score">0.9817981</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:10:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17347" length="10"/>
<text>CtGH5E279S</text>
</annotation>
<annotation id="445">
<infon key="score">0.8062502</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:51:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17358" length="4"/>
<text>CBM6</text>
</annotation>
<annotation id="446">
<infon key="score">0.9989459</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17368" length="9"/>
<text>cellulase</text>
</annotation>
<annotation id="447">
<infon key="score">0.999137</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:06Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17378" length="7"/>
<text>BaCel5A</text>
</annotation>
<annotation id="448">
<infon key="score">0.99901605</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:34:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17395" length="8"/>
<text>xylanase</text>
</annotation>
<annotation id="449">
<infon key="score">0.99872714</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17404" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="450">
<infon key="score">0.97036284</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:10:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17419" length="10"/>
<text>CtGH5E279S</text>
</annotation>
<annotation id="451">
<infon key="score">0.79618585</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:51:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17430" length="4"/>
<text>CBM6</text>
</annotation>
<annotation id="452">
<infon key="score">0.9977508</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17438" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="453">
<infon key="score">0.9992555</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:12Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17456" length="15"/>
<text>pentasaccharide</text>
</annotation>
<annotation id="454">
<infon key="score">0.99919766</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:14Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17473" length="17"/>
<text>β1,4-xylotetraose</text>
</annotation>
<annotation id="455">
<infon key="score">0.99904007</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17499" length="6"/>
<text>l-Araf</text>
</annotation>
<annotation id="456">
<infon key="score">0.9987269</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17538" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="457">
<infon key="score">0.9961971</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:20:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17591" length="16"/>
<text>hydrogen bonding</text>
</annotation>
<annotation id="458">
<infon key="score">0.99668086</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17616" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
<annotation id="459">
<infon key="score">0.9985068</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="17659" length="17"/>
<text>negative subsites</text>
</annotation>
<annotation id="460">
<infon key="score">0.9700168</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17681" length="7"/>
<text>density</text>
</annotation>
<annotation id="953">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:42Z</infon>
<location offset="17727" length="48"/>
<text>maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ</text>
</annotation>
<annotation id="461">
<infon key="score">0.999212</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:09Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17793" length="7"/>
<text>BaCel5A</text>
</annotation>
<annotation id="462">
<infon key="score">0.99365395</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17801" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="463">
<infon key="score">0.9992079</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17817" length="32"/>
<text>deoxy-2-fluoro-β-d-cellotrioside</text>
</annotation>
<annotation id="464">
<infon key="score">0.9983261</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17884" length="8"/>
<text>CmXyn10B</text>
</annotation>
<annotation id="894">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:04Z</infon>
<location offset="17893" length="15"/>
<text>in complex with</text>
</annotation>
<annotation id="465">
<infon key="score">0.9992083</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17911" length="10"/>
<text>xylotriose</text>
</annotation>
<annotation id="466">
<infon key="score">0.9557879</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:10:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18018" length="10"/>
<text>CtGH5E279S</text>
</annotation>
<annotation id="467">
<infon key="score">0.9193694</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:51:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18029" length="4"/>
<text>CBM6</text>
</annotation>
<annotation id="468">
<infon key="score">0.99914455</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:17Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18043" length="7"/>
<text>BaCel5A</text>
</annotation>
<annotation id="469">
<infon key="score">0.9989845</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:34:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18068" length="8"/>
<text>xylanase</text>
</annotation>
<annotation id="470">
<infon key="score">0.9984413</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18077" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="471">
<infon key="score">0.9951364</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18249" length="14"/>
<text>hydrogen bonds</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18283</offset>
<text>The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).</text>
<annotation id="472">
<infon key="score">0.99838185</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18299" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="473">
<infon key="score">0.99818134</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18338" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="474">
<infon key="score">0.7490116</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18344" length="2"/>
<text>CX</text>
</annotation>
<annotation id="475">
<infon key="score">0.9990195</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18397" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="476">
<infon key="score">0.9982736</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:48:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18412" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="477">
<infon key="score">0.99916756</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:30Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18476" length="12"/>
<text>xylotetraose</text>
</annotation>
<annotation id="478">
<infon key="score">0.9988993</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18528" length="11"/>
<text>active site</text>
</annotation>
<annotation id="479">
<infon key="score">0.998987</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18614" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="1019">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:33Z</infon>
<location offset="18630" length="17"/>
<text>subsites −2 to −4</text>
</annotation>
<annotation id="480">
<infon key="score">0.99836254</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:48:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18652" length="15"/>
<text>solvent-exposed</text>
</annotation>
<annotation id="481">
<infon key="score">0.9971443</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:41:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18721" length="8"/>
<text>pyranose</text>
</annotation>
<annotation id="482">
<infon key="score">0.846215</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18730" length="6"/>
<text>sugars</text>
</annotation>
<annotation id="1031">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:07:27Z</infon>
<location offset="18752" length="19"/>
<text>apolar interactions</text>
</annotation>
<annotation id="483">
<infon key="score">0.9933171</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18781" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="484">
<infon key="score">0.98697215</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18801" length="2"/>
<text>−2</text>
</annotation>
<annotation id="485">
<infon key="score">0.9984598</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18805" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="1030">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:07:11Z</infon>
<location offset="18816" length="26"/>
<text>planar apolar interactions</text>
</annotation>
<annotation id="486">
<infon key="score">0.99914765</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18852" length="4"/>
<text>Araf</text>
</annotation>
<annotation id="487">
<infon key="score">0.9965229</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18857" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="488">
<infon key="score">0.99492747</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:59:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18870" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="489">
<infon key="score">0.9988463</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18893" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="1020">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:00Z</infon>
<location offset="18901" length="18"/>
<text>subsites −2 and −3</text>
</annotation>
<annotation id="490">
<infon key="score">0.9847268</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18945" length="19"/>
<text>hydrophobic contact</text>
</annotation>
<annotation id="491">
<infon key="score">0.9995246</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18970" length="6"/>
<text>Val318</text>
</annotation>
<annotation id="492">
<infon key="score">0.8751849</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18982" length="2"/>
<text>−3</text>
</annotation>
<annotation id="493">
<infon key="score">0.9987112</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18985" length="4"/>
<text>Xylp</text>
</annotation>
<annotation id="1021">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:26Z</infon>
<location offset="18996" length="26"/>
<text>planar apolar interactions</text>
</annotation>
<annotation id="494">
<infon key="score">0.99950683</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19028" length="6"/>
<text>Ala137</text>
</annotation>
<annotation id="495">
<infon key="score">0.99882287</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19048" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="496">
<infon key="score">0.65771174</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19058" length="2"/>
<text>−4</text>
</annotation>
<annotation id="1029">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:50Z</infon>
<location offset="19067" length="24"/>
<text>parallel apolar contacts</text>
</annotation>
<annotation id="497">
<infon key="score">0.99949133</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19097" length="5"/>
<text>Trp69</text>
</annotation>
<annotation id="498">
<infon key="score">0.9983335</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19129" length="17"/>
<text>negative subsites</text>
</annotation>
<annotation id="499">
<infon key="score">0.9988159</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19150" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="500">
<infon key="score">0.9990707</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:23Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19163" length="7"/>
<text>BaCel5A</text>
</annotation>
<annotation id="501">
<infon key="score">0.9989895</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19185" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="502">
<infon key="score">0.9976179</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19190" length="8"/>
<text>xylanase</text>
</annotation>
<annotation id="503">
<infon key="score">0.9923934</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:27Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19200" length="8"/>
<text>CmXyn10B</text>
</annotation>
<annotation id="504">
<infon key="score">0.99495846</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19276" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="505">
<infon key="score">0.9988996</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19323" length="11"/>
<text>active site</text>
</annotation>
<annotation id="506">
<infon key="score">0.99886346</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19355" length="9"/>
<text>cellulase</text>
</annotation>
<annotation id="507">
<infon key="score">0.9983138</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19380" length="17"/>
<text>negative subsites</text>
</annotation>
<annotation id="508">
<infon key="score">0.98889786</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19406" length="6"/>
<text>sugars</text>
</annotation>
<annotation id="509">
<infon key="score">0.98305774</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19413" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="510">
<infon key="score">0.9832241</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19426" length="18"/>
<text>−2 and −3 subsites</text>
</annotation>
<annotation id="511">
<infon key="score">0.9674461</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19463" length="18"/>
<text>polar interactions</text>
</annotation>
<annotation id="512">
<infon key="score">0.99883157</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:31:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19521" length="4"/>
<text>GH10</text>
</annotation>
<annotation id="513">
<infon key="score">0.99209</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19526" length="8"/>
<text>xylanase</text>
</annotation>
<annotation id="514">
<infon key="score">0.99729645</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:00:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19551" length="10"/>
<text>−2 subsite</text>
</annotation>
<annotation id="515">
<infon key="score">0.99879384</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19583" length="9"/>
<text>cellulase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_4</infon>
<offset>19682</offset>
<text>The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity</text>
<annotation id="516">
<infon key="score">0.98137945</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19727" length="7"/>
<text>CtXyl5A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>19757</offset>
<text>CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.</text>
<annotation id="517">
<infon key="score">0.9990533</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19757" length="7"/>
<text>CtXyl5A</text>
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<annotation id="518">
<infon key="score">0.99842465</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19785" length="16"/>
<text>catalytic module</text>
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<annotation id="519">
<infon key="score">0.99872273</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19818" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="520">
<infon key="score">0.8389996</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19824" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="521">
<infon key="score">0.9042827</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19832" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="522">
<infon key="score">0.9175341</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19845" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="523">
<infon key="score">0.9989512</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19860" length="18"/>
<text>fibronectin domain</text>
</annotation>
<annotation id="524">
<infon key="score">0.99350643</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:45:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19880" length="5"/>
<text>CtFn3</text>
</annotation>
<annotation id="525">
<infon key="score">0.9994</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:51:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19930" length="4"/>
<text>CBM6</text>
</annotation>
<annotation id="526">
<infon key="score">0.93146616</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:33:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19935" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="527">
<infon key="score">0.9521939</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19947" length="8"/>
<text>exo-mode</text>
</annotation>
<annotation id="528">
<infon key="score">0.986774</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:44Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19959" length="33"/>
<text>xylo- and cellulooligosaccharides</text>
</annotation>
<annotation id="529">
<infon key="score">0.99714065</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20064" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="530">
<infon key="score">0.99528027</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20068" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="531">
<infon key="score">0.99527496</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20127" length="21"/>
<text>non-catalytic modules</text>
</annotation>
<annotation id="532">
<infon key="score">0.99911505</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20152" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="1032">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:08:05Z</infon>
<location offset="20198" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="533">
<infon key="score">0.99889565</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20227" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="534">
<infon key="score">0.96990776</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20288" length="10"/>
<text>removal of</text>
</annotation>
<annotation id="535">
<infon key="score">0.7631579</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20299" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="536">
<infon key="score">0.99899715</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20357" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="537">
<infon key="score">0.99888617</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20365" length="2"/>
<text>CX</text>
</annotation>
<annotation id="538">
<infon key="score">0.8609744</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20377" length="11"/>
<text>deletion of</text>
</annotation>
<annotation id="539">
<infon key="score">0.9990049</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20393" length="3"/>
<text>Fn3</text>
</annotation>
<annotation id="540">
<infon key="score">0.9974022</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20452" length="10"/>
<text>Truncation</text>
</annotation>
<annotation id="541">
<infon key="score">0.99084926</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20466" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="542">
<infon key="score">0.979066</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:52:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20560" length="5"/>
<text>CBM13</text>
</annotation>
<annotation id="543">
<infon key="score">0.9990688</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20593" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="544">
<infon key="score">0.99915814</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20601" length="7"/>
<text>mannose</text>
</annotation>
<annotation id="545">
<infon key="score">0.9989704</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20614" length="9"/>
<text>galactose</text>
</annotation>
<annotation id="546">
<infon key="score">0.777462</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20636" length="15"/>
<text>complex glycans</text>
</annotation>
<annotation id="547">
<infon key="score">0.9620865</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20682" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="548">
<infon key="score">0.99735713</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20739" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="549">
<infon key="score">0.9991768</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20759" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="550">
<infon key="score">0.9986268</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20768" length="15"/>
<text>Binding studies</text>
</annotation>
<annotation id="551">
<infon key="score">0.9760868</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20806" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="552">
<infon key="score">0.99847585</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:42:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20860" length="7"/>
<text>glycans</text>
</annotation>
<annotation id="553">
<infon key="score">0.99904245</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20878" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="554">
<infon key="score">0.99910945</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20883" length="2"/>
<text>CX</text>
</annotation>
<annotation id="555">
<infon key="score">0.999185</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20887" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="556">
<infon key="score">0.9991738</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:00Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20895" length="7"/>
<text>mannose</text>
</annotation>
<annotation id="557">
<infon key="score">0.99903405</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:03Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20904" length="9"/>
<text>galactose</text>
</annotation>
<annotation id="558">
<infon key="score">0.99697894</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20919" length="15"/>
<text>birchwood xylan</text>
</annotation>
<annotation id="559">
<infon key="score">0.9985886</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:07Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20936" length="2"/>
<text>BX</text>
</annotation>
<annotation id="560">
<infon key="score">0.9839033</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20991" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="561">
<infon key="score">0.99899536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21050" length="7"/>
<text>CtXyl5A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_4</infon>
<offset>21061</offset>
<text>Crystal Structure of CtXyl5A-D</text>
<annotation id="562">
<infon key="score">0.99862117</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21061" length="17"/>
<text>Crystal Structure</text>
</annotation>
<annotation id="563">
<infon key="score">0.9985922</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21082" length="9"/>
<text>CtXyl5A-D</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>21092</offset>
<text>To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously.</text>
<annotation id="564">
<infon key="score">0.99891555</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21127" length="21"/>
<text>non-catalytic modules</text>
</annotation>
<annotation id="565">
<infon key="score">0.99892277</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21152" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="566">
<infon key="score">0.99893296</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21164" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="567">
<infon key="score">0.9983997</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21185" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="568">
<infon key="score">0.7888786</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21208" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="569">
<infon key="score">0.4889835</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21217" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="570">
<infon key="score">0.94939154</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:05:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21285" length="12"/>
<text>crystallized</text>
</annotation>
<annotation id="571">
<infon key="score">0.9952478</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21325" length="7"/>
<text>without</text>
</annotation>
<annotation id="909">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:10Z</infon>
<location offset="21348" length="8"/>
<text>dockerin</text>
</annotation>
<annotation id="572">
<infon key="score">0.9989806</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21444" length="9"/>
<text>CtXyl5A-D</text>
</annotation>
<annotation id="573">
<infon key="score">0.9989059</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21459" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="574">
<infon key="score">0.9990243</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21484" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="575">
<infon key="score">0.99872184</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21518" length="21"/>
<text>molecular replacement</text>
</annotation>
<annotation id="576">
<infon key="score">0.9985116</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:28:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21571" length="5"/>
<text>Rwork</text>
</annotation>
<annotation id="577">
<infon key="score">0.99814725</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:28:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21581" length="5"/>
<text>Rfree</text>
</annotation>
<annotation id="578">
<infon key="score">0.99822944</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21625" length="9"/>
<text>structure</text>
</annotation>
<annotation id="579">
<infon key="score">0.967706</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:28:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21685" length="15"/>
<text>Ala36 to Trp742</text>
</annotation>
<annotation id="1045">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:31:31Z</infon>
<location offset="21725" length="18"/>
<text>GH5-CBM6-CBM13-Fn3</text>
</annotation>
<annotation id="580">
<infon key="score">0.998767</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21769" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="581">
<infon key="score">0.86748755</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21790" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="1022">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:01:19Z</infon>
<location offset="21892" length="15"/>
<text>crystal packing</text>
</annotation>
<annotation id="582">
<infon key="score">0.9981135</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:01:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21925" length="15"/>
<text>solvent channel</text>
</annotation>
<annotation id="583">
<infon key="score">0.61647874</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21966" length="5"/>
<text>CBM62</text>
</annotation>
<annotation id="584">
<infon key="score">0.99834853</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:28:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22024" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="585">
<infon key="score">0.85926026</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22055" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="586">
<infon key="score">0.8196963</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:36:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22069" length="6"/>
<text>mobile</text>
</annotation>
<annotation id="587">
<infon key="score">0.9980434</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22119" length="10"/>
<text>structures</text>
</annotation>
<annotation id="588">
<infon key="score">0.9983726</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22133" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="589">
<infon key="score">0.9979448</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22143" length="6"/>
<text>CtCBM6</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440005.jpg</infon>
<infon key="id">F5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>22184</offset>
<text>Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks.</text>
<annotation id="590">
<infon key="score">0.9992005</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22228" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="591">
<infon key="score">0.99799275</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22265" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="592">
<infon key="score">0.9989273</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22271" length="4"/>
<text>loop</text>
</annotation>
<annotation id="593">
<infon key="score">0.9980647</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22300" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="594">
<infon key="score">0.9991582</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22306" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="595">
<infon key="score">0.9944258</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22360" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="596">
<infon key="score">0.9965469</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22393" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="597">
<infon key="score">0.99827754</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22431" length="18"/>
<text>fibronectin domain</text>
</annotation>
<annotation id="598">
<infon key="score">0.99751484</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22536" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="599">
<infon key="score">0.99883205</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22542" length="4"/>
<text>loop</text>
</annotation>
<annotation id="600">
<infon key="score">0.9829196</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22573" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="601">
<infon key="score">0.9829951</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22588" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="602">
<infon key="score">0.99495596</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22636" length="14"/>
<text>hydrogen bonds</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>22746</offset>
<text>CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).</text>
<annotation id="603">
<infon key="score">0.9912047</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22746" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="604">
<infon key="score">0.79160744</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:29:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22767" length="16"/>
<text>Gly567 to Pro648</text>
</annotation>
<annotation id="940">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:29:04Z</infon>
<location offset="22796" length="5"/>
<text>CBM13</text>
</annotation>
<annotation id="605">
<infon key="score">0.97495085</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22811" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="606">
<infon key="score">0.99881685</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22830" length="14"/>
<text>β-trefoil fold</text>
</annotation>
<annotation id="607">
<infon key="score">0.99027336</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22900" length="21"/>
<text>3-fold repeating unit</text>
</annotation>
<annotation id="608">
<infon key="score">0.78913796</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:29:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22925" length="16"/>
<text>40–50 amino acid</text>
</annotation>
<annotation id="609">
<infon key="score">0.998422</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22973" length="17"/>
<text>Ricin superfamily</text>
</annotation>
<annotation id="610">
<infon key="score">0.8408495</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22997" length="6"/>
<text>repeat</text>
</annotation>
<annotation id="611">
<infon key="score">0.9983705</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23026" length="22"/>
<text>antiparallel β-strands</text>
</annotation>
<annotation id="612">
<infon key="score">0.9987341</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23052" length="11"/>
<text>Dali search</text>
</annotation>
<annotation id="941">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:29:39Z</infon>
<location offset="23102" length="5"/>
<text>CBM13</text>
</annotation>
<annotation id="613">
<infon key="score">0.998098</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:31:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23123" length="26"/>
<text>root mean square deviation</text>
</annotation>
<annotation id="614">
<infon key="score">0.99859744</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23255" length="15"/>
<text>C. thermocellum</text>
</annotation>
<annotation id="955">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:30:06Z</infon>
<location offset="23271" length="21"/>
<text>exo-β-1,3-galactanase</text>
</annotation>
<annotation id="615">
<infon key="score">0.9984226</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:30:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23310" length="24"/>
<text>Streptomyces avermitilis</text>
</annotation>
<annotation id="956">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:30:39Z</infon>
<location offset="23335" length="23"/>
<text>β-l-arabinopyranosidase</text>
</annotation>
<annotation id="616">
<infon key="score">0.9983331</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:30:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23376" length="21"/>
<text>Streptomyces lividans</text>
</annotation>
<annotation id="617">
<infon key="score">0.8736156</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:47:34Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23398" length="12"/>
<text>xylanase 10A</text>
</annotation>
<annotation id="1010">
<infon key="type">species</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:49:01Z</infon>
<location offset="23433" length="33"/>
<text>Streptomyces olivaceoviridis E-86</text>
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<annotation id="618">
<infon key="score">0.7191514</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:49:07Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23467" length="12"/>
<text>xylanase 10A</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>23509</offset>
<text>The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).</text>
<annotation id="906">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:38:04Z</infon>
<location offset="23513" length="3"/>
<text>Fn3</text>
</annotation>
<annotation id="619">
<infon key="score">0.9970993</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23543" length="15"/>
<text>β-sandwich fold</text>
</annotation>
<annotation id="1014">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:42Z</infon>
<location offset="23572" length="6"/>
<text>sheets</text>
</annotation>
<annotation id="620">
<infon key="score">0.9945593</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23608" length="20"/>
<text>antiparallel strands</text>
</annotation>
<annotation id="621">
<infon key="score">0.99866545</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23642" length="8"/>
<text>β1-β2-β5</text>
</annotation>
<annotation id="622">
<infon key="score">0.9991293</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23654" length="9"/>
<text>β-sheet 1</text>
</annotation>
<annotation id="623">
<infon key="score">0.9986183</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23668" length="8"/>
<text>β4-β3-β6</text>
</annotation>
<annotation id="624">
<infon key="score">0.99922734</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23680" length="9"/>
<text>β-sheet 2</text>
</annotation>
<annotation id="625">
<infon key="score">0.999304</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:32:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23700" length="9"/>
<text>β-sheet 2</text>
</annotation>
<annotation id="626">
<infon key="score">0.6592578</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:01:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23721" length="5"/>
<text>cleft</text>
</annotation>
<annotation id="627">
<infon key="score">0.732665</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:31:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23753" length="17"/>
<text>endo-binding CBMs</text>
</annotation>
<annotation id="628">
<infon key="score">0.9990554</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:07:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23878" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="629">
<infon key="score">0.66877615</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:49:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23890" length="6"/>
<text>enzyme</text>
</annotation>
<annotation id="630">
<infon key="score">0.9982305</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:01:38Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23902" length="5"/>
<text>cleft</text>
</annotation>
<annotation id="631">
<infon key="score">0.998874</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23919" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="632">
<infon key="score">0.9986582</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:28:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23981" length="14"/>
<text>polysaccharide</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>24045</offset>
<text>In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.</text>
<annotation id="633">
<infon key="score">0.99554837</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24052" length="9"/>
<text>structure</text>
</annotation>
<annotation id="634">
<infon key="score">0.99779385</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:27:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24065" length="9"/>
<text>CtXyl5A-D</text>
</annotation>
<annotation id="635">
<infon key="score">0.7459186</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24085" length="7"/>
<text>modules</text>
</annotation>
<annotation id="636">
<infon key="score">0.9990779</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:01:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24155" length="10"/>
<text>interfaces</text>
</annotation>
<annotation id="1046">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:32:00Z</infon>
<location offset="24169" length="16"/>
<text>CtGH5-CBM6-CBM13</text>
</annotation>
<annotation id="637">
<infon key="score">0.9977719</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24310" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="638">
<infon key="score">0.99530184</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24320" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="1023">
<infon key="type">site</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:02:09Z</infon>
<location offset="24355" length="30"/>
<text>apolar solvent-exposed surface</text>
</annotation>
<annotation id="639">
<infon key="score">0.9960426</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:26:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24441" length="18"/>
<text>polar interactions</text>
</annotation>
<annotation id="640">
<infon key="score">0.99697936</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24498" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="641">
<infon key="score">0.9962404</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:10:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24519" length="12"/>
<text>salt bridges</text>
</annotation>
<annotation id="1024">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:02:27Z</infon>
<location offset="24537" length="29"/>
<text>apolar and polar interactions</text>
</annotation>
<annotation id="642">
<infon key="score">0.9987067</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:30:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24666" length="20"/>
<text>glycoside hydrolases</text>
</annotation>
<annotation id="643">
<infon key="score">0.99762887</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24700" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="644">
<infon key="score">0.9972363</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24706" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="645">
<infon key="score">0.99929976</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24726" length="14"/>
<text>central domain</text>
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<annotation id="996">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:21Z</infon>
<location offset="24748" length="14"/>
<text>interacts with</text>
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<annotation id="646">
<infon key="score">0.9982716</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24763" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="647">
<infon key="score">0.9980007</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24770" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="648">
<infon key="score">0.998733</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:33:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24782" length="5"/>
<text>CtFn3</text>
</annotation>
<annotation id="649">
<infon key="score">0.99688613</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24804" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="650">
<infon key="score">0.99871945</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24907" length="7"/>
<text>compact</text>
</annotation>
<annotation id="651">
<infon key="score">0.99666494</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24915" length="14"/>
<text>heterotetramer</text>
</annotation>
<annotation id="652">
<infon key="score">0.9980378</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:33:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24951" length="22"/>
<text>CtCBM6-CBM13 interface</text>
</annotation>
<annotation id="653">
<infon key="score">0.9993907</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24979" length="6"/>
<text>linker</text>
</annotation>
<annotation id="654">
<infon key="score">0.99948835</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:06:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24987" length="9"/>
<text>SPISTGTIP</text>
</annotation>
<annotation id="655">
<infon key="score">0.9606576</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:52:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25014" length="7"/>
<text>modules</text>
</annotation>
<annotation id="656">
<infon key="score">0.99290574</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:08:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25038" length="6"/>
<text>Ser514</text>
</annotation>
<annotation id="657">
<infon key="score">0.9987361</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:08:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25048" length="6"/>
<text>Pro522</text>
</annotation>
<annotation id="658">
<infon key="score">0.88606876</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25065" length="18"/>
<text>fixed conformation</text>
</annotation>
<annotation id="659">
<infon key="score">0.9965569</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25150" length="3"/>
<text>Ile</text>
</annotation>
<annotation id="1026">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:03:28Z</infon>
<location offset="25178" length="15"/>
<text>apolar contacts</text>
</annotation>
<annotation id="660">
<infon key="score">0.9991703</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25205" length="6"/>
<text>linker</text>
</annotation>
<annotation id="661">
<infon key="score">0.9988801</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25229" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="662">
<infon key="score">0.99847704</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25301" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="663">
<infon key="score">0.99815255</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25319" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="664">
<infon key="score">0.99904627</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:33:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25362" length="4"/>
<text>loop</text>
</annotation>
<annotation id="665">
<infon key="score">0.99917275</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:33:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25375" length="3"/>
<text>β-7</text>
</annotation>
<annotation id="666">
<infon key="score">0.99806833</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25383" length="3"/>
<text>α-7</text>
</annotation>
<annotation id="667">
<infon key="score">0.9991961</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:33:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25388" length="6"/>
<text>loop 7</text>
</annotation>
<annotation id="668">
<infon key="score">0.9988186</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25399" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="994">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:05:50Z</infon>
<location offset="25429" length="17"/>
<text>trimodular clover</text>
</annotation>
<annotation id="669">
<infon key="score">0.5572531</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25560" length="7"/>
<text>modules</text>
</annotation>
<annotation id="670">
<infon key="score">0.99958736</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25571" length="6"/>
<text>Trp285</text>
</annotation>
<annotation id="1033">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:08:51Z</infon>
<location offset="25588" length="20"/>
<text>intercalated between</text>
</annotation>
<annotation id="671">
<infon key="score">0.99859947</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25617" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="672">
<infon key="score">0.997342</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:19:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25661" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="673">
<infon key="score">0.99960214</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25706" length="6"/>
<text>Val615</text>
</annotation>
<annotation id="674">
<infon key="score">0.9995946</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25717" length="6"/>
<text>Gly616</text>
</annotation>
<annotation id="675">
<infon key="score">0.9986933</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25727" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="1025">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:02:58Z</infon>
<location offset="25770" length="15"/>
<text>apolar contacts</text>
</annotation>
<annotation id="676">
<infon key="score">0.9936412</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25791" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="677">
<infon key="score">0.99960405</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25799" length="6"/>
<text>Pro440</text>
</annotation>
<annotation id="678">
<infon key="score">0.99960774</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25807" length="6"/>
<text>Phe489</text>
</annotation>
<annotation id="679">
<infon key="score">0.9996075</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25815" length="6"/>
<text>Gly491</text>
</annotation>
<annotation id="680">
<infon key="score">0.99959916</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25827" length="6"/>
<text>Ala492</text>
</annotation>
<annotation id="681">
<infon key="score">0.99917793</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25853" length="6"/>
<text>loop 7</text>
</annotation>
<annotation id="682">
<infon key="score">0.98367256</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25863" length="21"/>
<text>completely disordered</text>
</annotation>
<annotation id="683">
<infon key="score">0.9988607</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:37:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25892" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="684">
<infon key="score">0.69755924</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25916" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="685">
<infon key="score">0.9978527</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25935" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="686">
<infon key="score">0.9964631</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25945" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="687">
<infon key="score">0.99774665</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25994" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="688">
<infon key="score">0.9942677</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26037" length="4"/>
<text>loop</text>
</annotation>
<annotation id="689">
<infon key="score">0.9990918</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26063" length="6"/>
<text>loop 7</text>
</annotation>
<annotation id="690">
<infon key="score">0.9989008</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26122" length="11"/>
<text>active site</text>
</annotation>
<annotation id="691">
<infon key="score">0.9995603</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26183" length="6"/>
<text>Glu279</text>
</annotation>
<annotation id="692">
<infon key="score">0.9958968</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26221" length="4"/>
<text>loop</text>
</annotation>
<annotation id="693">
<infon key="score">0.5024981</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:34:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26239" length="7"/>
<text>removal</text>
</annotation>
<annotation id="694">
<infon key="score">0.99695325</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26250" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="1027">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:04:06Z</infon>
<location offset="26417" length="8"/>
<text>deletion</text>
</annotation>
<annotation id="904">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<location offset="26429" length="7"/>
<text>CtCBM13</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>26451</offset>
<text>Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.</text>
<annotation id="695">
<infon key="score">0.96206796</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26487" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="696">
<infon key="score">0.983107</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26498" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="697">
<infon key="score">0.99704546</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:25:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26527" length="24"/>
<text>hydrophobic interactions</text>
</annotation>
<annotation id="698">
<infon key="score">0.994769</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26560" length="7"/>
<text>CtCBM13</text>
</annotation>
<annotation id="699">
<infon key="score">0.99780685</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26572" length="5"/>
<text>CtFn3</text>
</annotation>
<annotation id="700">
<infon key="score">0.7309727</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26630" length="7"/>
<text>modules</text>
</annotation>
<annotation id="701">
<infon key="score">0.9990598</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:07:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26660" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="702">
<infon key="score">0.9950388</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26671" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="703">
<infon key="score">0.997883</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26686" length="9"/>
<text>structure</text>
</annotation>
<annotation id="704">
<infon key="score">0.9134587</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26714" length="6"/>
<text>module</text>
</annotation>
<annotation id="705">
<infon key="score">0.9861158</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:36Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26795" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="1028">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:05:12Z</infon>
<location offset="26807" length="10"/>
<text>binding to</text>
</annotation>
<annotation id="706">
<infon key="score">0.99920434</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26831" length="6"/>
<text>d-Galp</text>
</annotation>
<annotation id="707">
<infon key="score">0.99922657</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26842" length="6"/>
<text>l-Arap</text>
</annotation>
<annotation id="708">
<infon key="score">0.998511</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26853" length="5"/>
<text>plant</text>
</annotation>
<annotation id="709">
<infon key="score">0.99731666</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26932" length="6"/>
<text>Xylans</text>
</annotation>
<annotation id="710">
<infon key="score">0.9827069</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:11Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26981" length="6"/>
<text>sugars</text>
</annotation>
<annotation id="711">
<infon key="score">0.9991762</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26989" length="6"/>
<text>d-Galp</text>
</annotation>
<annotation id="712">
<infon key="score">0.99790454</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27027" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="713">
<infon key="score">0.99599224</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:43:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27056" length="6"/>
<text>cereal</text>
</annotation>
<annotation id="714">
<infon key="score">0.81789005</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27077" length="16"/>
<text>eucalyptus trees</text>
</annotation>
<annotation id="715">
<infon key="score">0.99418384</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27124" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="716">
<infon key="score">0.99656916</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27139" length="7"/>
<text>CtCBM62</text>
</annotation>
<annotation id="717">
<infon key="score">0.99837226</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27193" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="718">
<infon key="score">0.9991806</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:35:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27211" length="6"/>
<text>d-Galp</text>
</annotation>
<annotation id="719">
<infon key="score">0.9913663</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:06:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27286" length="4"/>
<text>open</text>
</annotation>
<annotation id="720">
<infon key="score">0.9990905</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27291" length="23"/>
<text>substrate binding cleft</text>
</annotation>
<annotation id="721">
<infon key="score">0.9991543</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27322" length="15"/>
<text>arabinoxylanase</text>
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<passage>
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<text>Exploring GH5 Subfamily 34</text>
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<text>CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.</text>
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<text>CtXyl5A</text>
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<infon key="updated_at">2023-09-18T10:45:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29470" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="763">
<infon key="score">0.9990416</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29479" length="2"/>
<text>CX</text>
</annotation>
<annotation id="764">
<infon key="score">0.9986411</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29511" length="2"/>
<text>BX</text>
</annotation>
<annotation id="765">
<infon key="score">0.9990243</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29559" length="16"/>
<text>arabinoxylanases</text>
</annotation>
<annotation id="766">
<infon key="score">0.98134685</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29609" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="767">
<infon key="score">0.9585189</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:50:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29618" length="5"/>
<text>AcGH5</text>
</annotation>
<annotation id="768">
<infon key="score">0.9965377</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29629" length="5"/>
<text>GpGH5</text>
</annotation>
<annotation id="769">
<infon key="score">0.9943236</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29656" length="16"/>
<text>oligosaccharides</text>
</annotation>
<annotation id="770">
<infon key="score">0.99477345</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T11:03:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29719" length="16"/>
<text>oligosaccharides</text>
</annotation>
<annotation id="771">
<infon key="score">0.9852911</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29790" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="772">
<infon key="score">0.9989858</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29863" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="773">
<infon key="score">0.9989502</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29910" length="16"/>
<text>arabinoxylanases</text>
</annotation>
<annotation id="977">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:42:40Z</infon>
<location offset="29939" length="7"/>
<text>GH43_10</text>
</annotation>
<annotation id="942">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:54:58Z</infon>
<location offset="29967" length="19"/>
<text>arabinofuranosidase</text>
</annotation>
<annotation id="981">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:42:23Z</infon>
<location offset="30000" length="4"/>
<text>GH43</text>
</annotation>
<annotation id="895">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<location offset="30010" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="932">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<location offset="30033" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="900">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<location offset="30076" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="937">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:50:32Z</infon>
<location offset="30135" length="5"/>
<text>AcGH5</text>
</annotation>
<annotation id="885">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<location offset="30171" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="914">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:24Z</infon>
<location offset="30192" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="917">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:28Z</infon>
<location offset="30200" length="3"/>
<text>RAX</text>
</annotation>
<annotation id="922">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<location offset="30239" length="2"/>
<text>CX</text>
</annotation>
<annotation id="928">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:27Z</infon>
<location offset="30284" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="933">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<location offset="30294" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="978">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:42:40Z</infon>
<location offset="30502" length="7"/>
<text>GH43_10</text>
</annotation>
<annotation id="896">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<location offset="30533" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="889">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<location offset="30564" length="13"/>
<text>arabinoxylans</text>
</annotation>
<annotation id="890">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<location offset="30619" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="984">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:44:44Z</infon>
<location offset="30711" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="982">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:44:04Z</infon>
<location offset="30737" length="5"/>
<text>Asp45</text>
</annotation>
<annotation id="979">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:45:12Z</infon>
<location offset="30751" length="7"/>
<text>GH43_10</text>
</annotation>
<annotation id="934">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<location offset="30769" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="985">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:44:57Z</infon>
<location offset="30779" length="16"/>
<text>substituted with</text>
</annotation>
<annotation id="983">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:44:24Z</infon>
<location offset="30796" length="7"/>
<text>alanine</text>
</annotation>
<annotation id="901">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<location offset="30843" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="927">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:23Z</infon>
<location offset="30865" length="4"/>
<text>D45A</text>
</annotation>
<annotation id="986">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:45:25Z</infon>
<location offset="30870" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="897">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<location offset="30893" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="943">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:54:58Z</infon>
<location offset="30923" length="19"/>
<text>arabinofuranosidase</text>
</annotation>
<annotation id="980">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:43:05Z</infon>
<location offset="30969" length="7"/>
<text>GH43_10</text>
</annotation>
<annotation id="929">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:27Z</infon>
<location offset="30991" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="987">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:45:51Z</infon>
<location offset="31022" length="8"/>
<text>kinetics</text>
</annotation>
<annotation id="968">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="31038" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="891">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<location offset="31045" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="902">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<location offset="31061" length="16"/>
<text>catalytic module</text>
</annotation>
<annotation id="886">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:31Z</infon>
<location offset="31178" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="974">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:38:21Z</infon>
<location offset="31206" length="6"/>
<text>fungal</text>
</annotation>
<annotation id="892">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<location offset="31213" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="915">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:24Z</infon>
<location offset="31269" length="3"/>
<text>WAX</text>
</annotation>
<annotation id="918">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:45:28Z</infon>
<location offset="31277" length="3"/>
<text>RAX</text>
</annotation>
<annotation id="887">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:32Z</infon>
<location offset="31324" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="988">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:46:09Z</infon>
<location offset="31402" length="10"/>
<text>eukaryotic</text>
</annotation>
<annotation id="989">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:46:19Z</infon>
<location offset="31417" length="11"/>
<text>prokaryotic</text>
</annotation>
<annotation id="923">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<location offset="31445" length="2"/>
<text>CX</text>
</annotation>
<annotation id="938">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:50:32Z</infon>
<location offset="31519" length="5"/>
<text>AcGH5</text>
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<annotation id="935">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<location offset="31529" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="995">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:07:19Z</infon>
<location offset="31651" length="11"/>
<text>full-length</text>
</annotation>
</passage>
<passage>
<infon key="file">zbc0441653440006.jpg</infon>
<infon key="id">F6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>31787</offset>
<text>Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.</text>
<annotation id="969">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="31817" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="774">
<infon key="score">0.93129015</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:46:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31858" length="9"/>
<text>incubated</text>
</annotation>
<annotation id="775">
<infon key="score">0.99875844</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="31892" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="776">
<infon key="score">0.9989768</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31957" length="5"/>
<text>GpGH5</text>
</annotation>
<annotation id="777">
<infon key="score">0.9987948</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31964" length="5"/>
<text>VbGH5</text>
</annotation>
<annotation id="778">
<infon key="score">0.9988304</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:50:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31975" length="5"/>
<text>AcGH5</text>
</annotation>
<annotation id="779">
<infon key="score">0.9960063</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="32029" length="3"/>
<text>TLC</text>
</annotation>
<annotation id="780">
<infon key="score">0.99918824</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32038" length="19"/>
<text>xylooligosaccharide</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>32125</offset>
<text>Discussion</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>32136</offset>
<text>A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.</text>
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<infon key="updated_at">2023-09-18T10:24:42Z</infon>
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<text>plant</text>
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<annotation id="782">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32249" length="4"/>
<text>CBMs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32340" length="17"/>
<text>catalytic modules</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:30Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32366" length="25"/>
<text>flexible linker sequences</text>
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<annotation id="785">
<infon key="score">0.5641079</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32393" length="7"/>
<text>CtXyl5A</text>
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<annotation id="786">
<infon key="score">0.99627954</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32438" length="9"/>
<text>structure</text>
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<annotation id="787">
<infon key="score">0.99776864</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32570" length="4"/>
<text>CBMs</text>
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<annotation id="788">
<infon key="score">0.86189497</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32576" length="6"/>
<text>CtCBM6</text>
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<annotation id="789">
<infon key="score">0.95701545</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32587" length="7"/>
<text>CtCBM13</text>
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<annotation id="790">
<infon key="score">0.99906987</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32615" length="6"/>
<text>active</text>
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<annotation id="791">
<infon key="score">0.9981731</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32642" length="16"/>
<text>catalytic module</text>
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<annotation id="792">
<infon key="score">0.9962853</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32660" length="5"/>
<text>CtGH5</text>
</annotation>
<annotation id="793">
<infon key="score">0.99705863</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:51:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32671" length="21"/>
<text>crystallographic data</text>
</annotation>
<annotation id="794">
<infon key="score">0.998901</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32795" length="7"/>
<text>glycans</text>
</annotation>
<annotation id="795">
<infon key="score">0.9889486</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:37:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32804" length="7"/>
<text>CtCBM13</text>
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<annotation id="796">
<infon key="score">0.99855596</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32864" length="5"/>
<text>xylan</text>
</annotation>
<annotation id="797">
<infon key="score">0.99709713</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32873" length="9"/>
<text>cellulose</text>
</annotation>
<annotation id="798">
<infon key="score">0.9898572</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32891" length="6"/>
<text>CtCBM6</text>
</annotation>
<annotation id="799">
<infon key="score">0.9853876</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:43:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32982" length="6"/>
<text>glycan</text>
</annotation>
<annotation id="800">
<infon key="score">0.99907523</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:07:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33048" length="11"/>
<text>full-length</text>
</annotation>
<annotation id="801">
<infon key="score">0.9986217</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33115" length="5"/>
<text>plant</text>
</annotation>
<annotation id="802">
<infon key="score">0.9963482</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33158" length="5"/>
<text>CBM46</text>
</annotation>
<annotation id="803">
<infon key="score">0.9411532</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:23Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33178" length="8"/>
<text>Bacillus</text>
</annotation>
<annotation id="804">
<infon key="score">0.99831474</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33187" length="13"/>
<text>xyloglucanase</text>
</annotation>
<annotation id="805">
<infon key="score">0.9972825</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33201" length="22"/>
<text>mixed linked glucanase</text>
</annotation>
<annotation id="806">
<infon key="score">0.9992636</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:30Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33224" length="7"/>
<text>BhCel5B</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>33233</offset>
<text>CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.</text>
<annotation id="807">
<infon key="score">0.9979913</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33233" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="808">
<infon key="score">0.94887006</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33256" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="809">
<infon key="score">0.9977837</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33374" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="970">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="33407" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="810">
<infon key="score">0.99929583</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="33581" length="16"/>
<text>arabinoxylanases</text>
</annotation>
<annotation id="971">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="33667" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="811">
<infon key="score">0.99110305</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:04:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33688" length="24"/>
<text>specificity determinants</text>
</annotation>
<annotation id="812">
<infon key="score">0.99954575</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33713" length="5"/>
<text>Glu68</text>
</annotation>
<annotation id="813">
<infon key="score">0.9995704</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33720" length="5"/>
<text>Tyr92</text>
</annotation>
<annotation id="814">
<infon key="score">0.99954885</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33731" length="6"/>
<text>Asn139</text>
</annotation>
<annotation id="815">
<infon key="score">0.9985304</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="33781" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="816">
<infon key="score">0.99864835</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="33791" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="817">
<infon key="score">0.9986283</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:04:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33808" length="11"/>
<text>−2* subsite</text>
</annotation>
<annotation id="818">
<infon key="score">0.9983754</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="33849" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="819">
<infon key="score">0.99908876</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:27:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33874" length="11"/>
<text>active site</text>
</annotation>
<annotation id="820">
<infon key="score">0.99881303</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33905" length="5"/>
<text>CBM62</text>
</annotation>
<annotation id="821">
<infon key="score">0.9981012</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33914" length="7"/>
<text>CtXyl5A</text>
</annotation>
<annotation id="822">
<infon key="score">0.9863192</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:50:32Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33926" length="5"/>
<text>AcGH5</text>
</annotation>
<annotation id="823">
<infon key="score">0.99910295</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="33982" length="6"/>
<text>xylans</text>
</annotation>
<annotation id="824">
<infon key="score">0.9990118</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:44:04Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34002" length="11"/>
<text>d-galactose</text>
</annotation>
<annotation id="825">
<infon key="score">0.70076585</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:07:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="34040" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="826">
<infon key="score">0.9968041</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="34070" length="3"/>
<text>CBM</text>
</annotation>
<annotation id="827">
<infon key="score">0.9990013</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:47:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="34077" length="5"/>
<text>GpGH5</text>
</annotation>
<annotation id="828">
<infon key="score">0.99921906</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34106" length="15"/>
<text>arabinoxylanase</text>
</annotation>
<annotation id="829">
<infon key="score">0.9991093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34152" length="13"/>
<text>arabinoxylans</text>
</annotation>
<annotation id="830">
<infon key="score">0.9977469</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:44:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="34194" length="7"/>
<text>cereals</text>
</annotation>
<annotation id="972">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="34251" length="6"/>
<text>GH5_34</text>
</annotation>
<annotation id="831">
<infon key="score">0.9990747</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34368" length="3"/>
<text>GHs</text>
</annotation>
<annotation id="832">
<infon key="score">0.97536546</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:49:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34382" length="4"/>
<text>GH43</text>
</annotation>
<annotation id="833">
<infon key="score">0.993919</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:40:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34391" length="3"/>
<text>GH5</text>
</annotation>
<annotation id="973">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:36:54Z</infon>
<location offset="34420" length="6"/>
<text>GH5_34</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>34568</offset>
<text>An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).</text>
<annotation id="834">
<infon key="score">0.9990096</infon>
<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="34593" length="5"/>
<text>VbGH5</text>
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<annotation id="835">
<infon key="score">0.9992551</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="34703" length="16"/>
<text>arabinoxylanases</text>
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<annotation id="836">
<infon key="score">0.9983157</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34749" length="9"/>
<text>arabinose</text>
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<annotation id="837">
<infon key="score">0.999131</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:49:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="34805" length="5"/>
<text>VbGH5</text>
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<annotation id="838">
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34828" length="6"/>
<text>xylans</text>
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<annotation id="839">
<infon key="score">0.9986557</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34843" length="9"/>
<text>arabinose</text>
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<annotation id="840">
<infon key="score">0.9989998</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:25:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="34948" length="13"/>
<text>arabinoxylans</text>
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<annotation id="841">
<infon key="score">0.99906427</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35013" length="4"/>
<text>GH98</text>
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<annotation id="842">
<infon key="score">0.9987771</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:35:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35018" length="8"/>
<text>xylanase</text>
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<annotation id="843">
<infon key="score">0.9987264</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:33Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="35134" length="6"/>
<text>xylans</text>
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<annotation id="924">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:46:39Z</infon>
<location offset="35147" length="2"/>
<text>CX</text>
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</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>35152</offset>
<text>To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.</text>
<annotation id="844">
<infon key="score">0.9992999</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35238" length="16"/>
<text>arabinoxylanases</text>
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<annotation id="845">
<infon key="score">0.99896705</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:05:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="35298" length="6"/>
<text>pocket</text>
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<annotation id="846">
<infon key="score">0.99837506</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:42Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="35323" length="9"/>
<text>arabinose</text>
</annotation>
<annotation id="847">
<infon key="score">0.99813867</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:26:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="35336" length="6"/>
<text>xylose</text>
</annotation>
<annotation id="848">
<infon key="score">0.9551583</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:38:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="35360" length="4"/>
<text>open</text>
</annotation>
<annotation id="849">
<infon key="score">0.99909574</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:49:02Z</infon>
<infon key="identifier">SO:</infon>
<location offset="35365" length="19"/>
<text>xylan binding cleft</text>
</annotation>
<annotation id="850">
<infon key="score">0.9953093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:29:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="35457" length="13"/>
<text>hemicellulose</text>
</annotation>
<annotation id="851">
<infon key="score">0.9972641</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:53:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="35517" length="17"/>
<text>catalytic modules</text>
</annotation>
<annotation id="852">
<infon key="score">0.99897206</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:36:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="35539" length="4"/>
<text>CBMs</text>
</annotation>
<annotation id="853">
<infon key="score">0.9993069</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35604" length="16"/>
<text>arabinoxylanases</text>
</annotation>
<annotation id="854">
<infon key="score">0.9992654</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35696" length="16"/>
<text>arabinoxylanases</text>
</annotation>
<annotation id="855">
<infon key="score">0.9987754</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:35:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="35760" length="14"/>
<text>endo-xylanases</text>
</annotation>
<annotation id="856">
<infon key="score">0.9984994</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="35888" length="5"/>
<text>plant</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>35932</offset>
<text>Experimental Procedures</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_4</infon>
<offset>35956</offset>
<text>Cloning, Expression, and Purification of Components of CtXyl5A</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>36019</offset>
<text>All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>37237</offset>
<text>To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to >90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_4</infon>
<offset>38285</offset>
<text>Enzyme Assays</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>38299</offset>
<text>CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_4</infon>
<offset>39304</offset>
<text>Oligosaccharide Analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>39329</offset>
<text>Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously </text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_4</infon>
<offset>39853</offset>
<text>Crystallography</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>39869</offset>
<text>Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>41279</offset>
<text>To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2.</text>
</passage>
<passage>
<infon key="file">T2.xml</infon>
<infon key="id">T2</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>43158</offset>
<text>Data collection and refinement statistics</text>
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<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T12:51:14Z</infon>
<location offset="43158" length="41"/>
<text>Data collection and refinement statistics</text>
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<passage>
<infon key="file">T2.xml</infon>
<infon key="id">T2</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table_caption</infon>
<offset>43200</offset>
<text>The values in parentheses are for highest resolution shell.</text>
</passage>
<passage>
<infon key="file">T2.xml</infon>
<infon key="id">T2</infon>
<infon key="section_type">TABLE</infon>
<infon key="type">table</infon>
<infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead valign="bottom"><tr><th rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">CtXyl5A<sub>-D</sub></th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Arap</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Xylp</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6- (<italic>Araf</italic>-Xyl<italic>p</italic><sub>4</sub>)</th></tr></thead><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data collection</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Source</td><td align="left" rowspan="1" colspan="1">ESRF-ID14-1</td><td align="left" rowspan="1" colspan="1">Diamond I04–1</td><td align="left" rowspan="1" colspan="1">Diamond I24</td><td align="left" rowspan="1" colspan="1">Diamond I02</td></tr><tr><td align="left" rowspan="1" colspan="1"> Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9334</td><td align="left" rowspan="1" colspan="1">0.9173</td><td align="left" rowspan="1" colspan="1">0.9772</td><td align="left" rowspan="1" colspan="1">0.9791</td></tr><tr><td align="left" rowspan="1" colspan="1"> Space group</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" rowspan="1" colspan="1"> Cell dimensions</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">147.4, 191.7, 50.7</td><td align="left" rowspan="1" colspan="1">67.1, 72.4, 109.1</td><td align="left" rowspan="1" colspan="1">67.9, 72.5, 109.5</td><td align="left" rowspan="1" colspan="1">76.3, 123.2, 125.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> α, β, γ (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. of measured reflections</td><td align="left" rowspan="1" colspan="1">244,475 (29,324)</td><td align="left" rowspan="1" colspan="1">224,842 (11,281)</td><td align="left" rowspan="1" colspan="1">152,004 (4,996)</td><td align="left" rowspan="1" colspan="1">463,237 (23,068)</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. of independent reflections</td><td align="left" rowspan="1" colspan="1">42246 (5,920)</td><td align="left" rowspan="1" colspan="1">63,523 (3,175)</td><td align="left" rowspan="1" colspan="1">42,716 (2,334)</td><td align="left" rowspan="1" colspan="1">140,288 (6,879)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution (Å)</td><td align="left" rowspan="1" colspan="1">50.70–2.64 (2.78–2.64)</td><td align="left" rowspan="1" colspan="1">44.85–1.65 (1.68–1.65)</td><td align="left" rowspan="1" colspan="1">45.16–1.90 (1.94–1.90)</td><td align="left" rowspan="1" colspan="1">48.43–1.65 (1.68–1.65)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>merge</sub> (%)</td><td align="left" rowspan="1" colspan="1">16.5 (69.5)</td><td align="left" rowspan="1" colspan="1">6.7 (65.1)</td><td align="left" rowspan="1" colspan="1">2.8 (8.4)</td><td align="left" rowspan="1" colspan="1">5.7 (74.9)</td></tr><tr><td align="left" rowspan="1" colspan="1"> CC<sub>1/2</sub></td><td align="left" rowspan="1" colspan="1">0.985 (0.478)</td><td align="left" rowspan="1" colspan="1">0.998 (0.594)</td><td align="left" rowspan="1" colspan="1">0.999 (0.982)</td><td align="left" rowspan="1" colspan="1">0.998 (0.484)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>I</italic>/σ<italic>I</italic></td><td align="left" rowspan="1" colspan="1">8.0 (2.0)</td><td align="left" rowspan="1" colspan="1">13 (1.6)</td><td align="left" rowspan="1" colspan="1">26.6 (8.0)</td><td align="left" rowspan="1" colspan="1">11.2 (1.6)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Completeness (%)</td><td align="left" rowspan="1" colspan="1">98.5 (96.4)</td><td align="left" rowspan="1" colspan="1">98.5 (99.4)</td><td align="left" rowspan="1" colspan="1">98.6 (85.0)</td><td align="left" rowspan="1" colspan="1">98.8 (99.4)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Redundancy</td><td align="left" rowspan="1" colspan="1">5.8 (5.0)</td><td align="left" rowspan="1" colspan="1">3.5 (3.6)</td><td align="left" rowspan="1" colspan="1">3.6 (2.1)</td><td align="left" rowspan="1" colspan="1">3.3 (3.4)</td></tr><tr><td colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Refinement</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="left" rowspan="1" colspan="1">23.7/27.8</td><td align="left" rowspan="1" colspan="1">12.2/17.0</td><td align="left" rowspan="1" colspan="1">12.9/16.1</td><td align="left" rowspan="1" colspan="1">14.5/19.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. atoms</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Protein</td><td align="left" rowspan="1" colspan="1">5446</td><td align="left" rowspan="1" colspan="1">3790</td><td align="left" rowspan="1" colspan="1">3729</td><td align="left" rowspan="1" colspan="1">7333</td></tr><tr><td align="left" rowspan="1" colspan="1"> Ligand</td><td align="left" rowspan="1" colspan="1">19</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">92</td></tr><tr><td align="left" rowspan="1" colspan="1"> Water</td><td align="left" rowspan="1" colspan="1">227</td><td align="left" rowspan="1" colspan="1">579</td><td align="left" rowspan="1" colspan="1">601</td><td align="left" rowspan="1" colspan="1">923</td></tr><tr><td align="left" rowspan="1" colspan="1"> B-factors</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Protein</td><td align="left" rowspan="1" colspan="1">41.6</td><td align="left" rowspan="1" colspan="1">17.8</td><td align="left" rowspan="1" colspan="1">15.8</td><td align="left" rowspan="1" colspan="1">21.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Ligand</td><td align="left" rowspan="1" colspan="1">65.0</td><td align="left" rowspan="1" colspan="1">19.4</td><td align="left" rowspan="1" colspan="1">24.2</td><td align="left" rowspan="1" colspan="1">39.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> Water</td><td align="left" rowspan="1" colspan="1">35.4</td><td align="left" rowspan="1" colspan="1">38.5</td><td align="left" rowspan="1" colspan="1">32.2</td><td align="left" rowspan="1" colspan="1">37.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> R.m.s deviations</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.008</td><td align="left" rowspan="1" colspan="1">0.015</td><td align="left" rowspan="1" colspan="1">0.012</td><td align="left" rowspan="1" colspan="1">0.012</td></tr><tr><td align="left" rowspan="1" colspan="1"> Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.233</td><td align="left" rowspan="1" colspan="1">1.502</td><td align="left" rowspan="1" colspan="1">1.624</td><td align="left" rowspan="1" colspan="1">1.554</td></tr><tr><td align="left" rowspan="1" colspan="1"> Protein Data Bank code</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5G56">5G56</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA0">5LA0</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA1">5LA1</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="2LA2">2LA2</ext-link></td></tr></tbody></table>
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<text> CtXyl5A-D GH5-CBM6-Arap GH5-CBM6-Xylp GH5-CBM6- (Araf-Xylp4) Data collection Source ESRF-ID14-1 Diamond I04–1 Diamond I24 Diamond I02 Wavelength (Å) 0.9334 0.9173 0.9772 0.9791 Space group P21212 P212121 P212121 P212121 Cell dimensions a, b, c (Å) 147.4, 191.7, 50.7 67.1, 72.4, 109.1 67.9, 72.5, 109.5 76.3, 123.2, 125.4 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 No. of measured reflections 244,475 (29,324) 224,842 (11,281) 152,004 (4,996) 463,237 (23,068) No. of independent reflections 42246 (5,920) 63,523 (3,175) 42,716 (2,334) 140,288 (6,879) Resolution (Å) 50.70–2.64 (2.78–2.64) 44.85–1.65 (1.68–1.65) 45.16–1.90 (1.94–1.90) 48.43–1.65 (1.68–1.65) Rmerge (%) 16.5 (69.5) 6.7 (65.1) 2.8 (8.4) 5.7 (74.9) CC1/2 0.985 (0.478) 0.998 (0.594) 0.999 (0.982) 0.998 (0.484) I/σI 8.0 (2.0) 13 (1.6) 26.6 (8.0) 11.2 (1.6) Completeness (%) 98.5 (96.4) 98.5 (99.4) 98.6 (85.0) 98.8 (99.4) Redundancy 5.8 (5.0) 3.5 (3.6) 3.6 (2.1) 3.3 (3.4) Refinement Rwork/Rfree 23.7/27.8 12.2/17.0 12.9/16.1 14.5/19.9 No. atoms Protein 5446 3790 3729 7333 Ligand 19 20 20 92 Water 227 579 601 923 B-factors Protein 41.6 17.8 15.8 21.0 Ligand 65.0 19.4 24.2 39.5 Water 35.4 38.5 32.2 37.6 R.m.s deviations Bond lengths (Å) 0.008 0.015 0.012 0.012 Bond angles (°) 1.233 1.502 1.624 1.554 Protein Data Bank code 5G56 5LA0 5LA1 2LA2 </text>
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<text>GH5-CBM6- (Araf-Xylp4)</text>
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<text>Rwork</text>
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<passage>
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<text>Author Contributions</text>
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<infon key="type">paragraph</infon>
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<text>A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper.</text>
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<text>Supplementary Material</text>
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<infon key="type">footnote</infon>
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<text>This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article.</text>
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<text>This article contains supplemental Table S1 and Fig. S1.</text>
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<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46740" length="5"/>
<text>xylan</text>
</annotation>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46746</offset>
<text>HPAEC</text>
<annotation id="879">
<infon key="score">0.8287563</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:58:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="46746" length="5"/>
<text>HPAEC</text>
</annotation>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46752</offset>
<text>high performance anion exchange chromatography</text>
<annotation id="880">
<infon key="score">0.99853724</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="46752" length="46"/>
<text>high performance anion exchange chromatography</text>
</annotation>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46799</offset>
<text>PDB</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46803</offset>
<text>Protein Data Bank</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46821</offset>
<text>BX</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46824</offset>
<text>birchwood xylan</text>
<annotation id="881">
<infon key="score">0.96442974</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T13:44:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="46824" length="9"/>
<text>birchwood</text>
</annotation>
<annotation id="882">
<infon key="score">0.99584645</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:24:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="46834" length="5"/>
<text>xylan</text>
</annotation>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46840</offset>
<text>ESI</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>46844</offset>
<text>electrospray ionization.</text>
<annotation id="883">
<infon key="score">0.99849916</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T14:06:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="46844" length="23"/>
<text>electrospray ionization</text>
</annotation>
</passage>
<passage>
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<infon key="type">footnote</infon>
<offset>46869</offset>
<text>The abbreviations used are: </text>
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<infon key="article-id_doi">10.18632/oncotarget.9692</infon>
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<infon key="kwd">DNA N6-adenine methyltransferase M1.HpyAVI substrate recognition AdoMet-binding Helicobacter pylori Immunology and Microbiology Section Immune response Immunity</infon>
<infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon>
<infon key="lpage">40977</infon>
<infon key="name_0">surname:Ma;given-names:Bo</infon>
<infon key="name_1">surname:Ma;given-names:Ji</infon>
<infon key="name_2">surname:Liu;given-names:Dong</infon>
<infon key="name_3">surname:Guo;given-names:Ling</infon>
<infon key="name_4">surname:Chen;given-names:Huiling</infon>
<infon key="name_5">surname:Ding;given-names:Jingjin</infon>
<infon key="name_6">surname:Liu;given-names:Wei</infon>
<infon key="name_7">surname:Zhang;given-names:Hongquan</infon>
<infon key="section_type">TITLE</infon>
<infon key="type">front</infon>
<infon key="volume">7</infon>
<infon key="year">2016</infon>
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<text>Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori </text>
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<text>DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.</text>
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<text>INTRODUCTION</text>
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<text>DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.</text>
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<text>Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.</text>
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<infon key="score">0.9946444</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2104" length="5"/>
<text>human</text>
</annotation>
<annotation id="35">
<infon key="score">0.9984837</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:52:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2171" length="11"/>
<text>prokaryotic</text>
</annotation>
<annotation id="36">
<infon key="score">0.9975991</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:52:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2183" length="18"/>
<text>DNA cytosine MTase</text>
</annotation>
<annotation id="37">
<infon key="score">0.998495</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:52:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2218" length="18"/>
<text>N4-cytosine MTases</text>
</annotation>
<annotation id="38">
<infon key="score">0.9925321</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2270" length="12"/>
<text>thermophilic</text>
</annotation>
<annotation id="39">
<infon key="score">0.9845115</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2286" length="10"/>
<text>mesophilic</text>
</annotation>
<annotation id="40">
<infon key="score">0.99733055</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:48:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2297" length="8"/>
<text>bacteria</text>
</annotation>
<annotation id="851">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="2318" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="41">
<infon key="score">0.662408</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2363" length="8"/>
<text>cytosine</text>
</annotation>
<annotation id="42">
<infon key="score">0.92875</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2373" length="3"/>
<text>4mC</text>
</annotation>
<annotation id="879">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:38Z</infon>
<location offset="2379" length="14"/>
<text>N4 methylation</text>
</annotation>
<annotation id="43">
<infon key="score">0.99867094</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2431" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="687">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:14:44Z</infon>
<location offset="2483" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="677">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:28Z</infon>
<location offset="2521" length="14"/>
<text>bacteriophages</text>
</annotation>
<annotation id="44">
<infon key="score">0.99827415</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2554" length="17"/>
<text>N6-adenine MTases</text>
</annotation>
<annotation id="45">
<infon key="score">0.9711873</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2612" length="7"/>
<text>adenine</text>
</annotation>
<annotation id="46">
<infon key="score">0.98714834</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2621" length="3"/>
<text>6mA</text>
</annotation>
<annotation id="47">
<infon key="score">0.9985636</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2643" length="11"/>
<text>prokaryotes</text>
</annotation>
<annotation id="688">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:14:54Z</infon>
<location offset="2687" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="48">
<infon key="score">0.96919215</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2883" length="3"/>
<text>6mA</text>
</annotation>
<annotation id="49">
<infon key="score">0.99854434</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2898" length="10"/>
<text>eukaryotic</text>
</annotation>
<annotation id="820">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:33:46Z</infon>
<location offset="2918" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="50">
<infon key="score">0.9743464</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2922" length="3"/>
<text>6mA</text>
</annotation>
<annotation id="51">
<infon key="score">0.84729904</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3065" length="13"/>
<text>Chlamydomonas</text>
</annotation>
<annotation id="52">
<infon key="score">0.99722385</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3125" length="9"/>
<text>C.elegans</text>
</annotation>
<annotation id="53">
<infon key="score">0.9626723</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3165" length="10"/>
<text>Drosophila</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3177</offset>
<text>All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.</text>
<annotation id="815">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:49Z</infon>
<location offset="3200" length="11"/>
<text>methylation</text>
</annotation>
<annotation id="54">
<infon key="score">0.99844944</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3221" length="11"/>
<text>prokaryotes</text>
</annotation>
<annotation id="55">
<infon key="score">0.99861634</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3243" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="56">
<infon key="score">0.9980486</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3409" length="24"/>
<text>restriction endonuclease</text>
</annotation>
<annotation id="689">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:15:09Z</infon>
<location offset="3452" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="57">
<infon key="score">0.99867034</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3490" length="30"/>
<text>modification methyltransferase</text>
</annotation>
<annotation id="58">
<infon key="score">0.99824834</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:48:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3649" length="8"/>
<text>bacteria</text>
</annotation>
<annotation id="59">
<infon key="score">0.99335164</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3676" length="14"/>
<text>bacteriophages</text>
</annotation>
<annotation id="690">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:15:18Z</infon>
<location offset="3791" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="60">
<infon key="score">0.99397075</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3855" length="41"/>
<text>DNA adenine or cytosine methyltransferase</text>
</annotation>
<annotation id="61">
<infon key="score">0.99784696</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4049" length="24"/>
<text>restriction endonuclease</text>
</annotation>
<annotation id="62">
<infon key="score">0.9003789</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:56:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4080" length="41"/>
<text>DNA adenine or cytosine methyltransferase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>4158</offset>
<text>To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.</text>
<annotation id="63">
<infon key="score">0.9985929</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4179" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="64">
<infon key="score">0.9986365</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4189" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="65">
<infon key="score">0.9897859</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4210" length="26"/>
<text>structurally characterized</text>
</annotation>
<annotation id="66">
<infon key="score">0.9991573</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4293" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="67">
<infon key="score">0.9947482</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4403" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="828">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:36:18Z</infon>
<location offset="4420" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="68">
<infon key="score">0.9982283</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4451" length="11"/>
<text>active site</text>
</annotation>
<annotation id="852">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="4467" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="69">
<infon key="score">0.9962698</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4498" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="821">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:34:46Z</infon>
<location offset="4528" length="31"/>
<text>target (DNA)-recognition domain</text>
</annotation>
<annotation id="733">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="4561" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="691">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:15:33Z</infon>
<location offset="4626" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="692">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:15:43Z</infon>
<location offset="4670" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="70">
<infon key="score">0.9656195</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4699" length="9"/>
<text>Conserved</text>
</annotation>
<annotation id="71">
<infon key="score">0.99657625</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:58:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4762" length="10"/>
<text>structures</text>
</annotation>
<annotation id="824">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:35:22Z</infon>
<location offset="4796" length="3"/>
<text>I-X</text>
</annotation>
<annotation id="72">
<infon key="score">0.99850804</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:58:34Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4804" length="15"/>
<text>cytosine MTases</text>
</annotation>
<annotation id="822">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:35:02Z</infon>
<location offset="4837" length="6"/>
<text>I-VIII</text>
</annotation>
<annotation id="823">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:35:13Z</infon>
<location offset="4848" length="1"/>
<text>X</text>
</annotation>
<annotation id="73">
<infon key="score">0.9986151</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:58:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4854" length="14"/>
<text>adenine MTases</text>
</annotation>
<annotation id="74">
<infon key="score">0.8387143</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4970" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="75">
<infon key="score">0.9985342</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:58:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4989" length="22"/>
<text>exocyclic amino MTases</text>
</annotation>
<annotation id="76">
<infon key="score">0.99915075</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5051" length="1"/>
<text>α</text>
</annotation>
<annotation id="77">
<infon key="score">0.9991696</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5054" length="1"/>
<text>β</text>
</annotation>
<annotation id="78">
<infon key="score">0.9992083</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5057" length="1"/>
<text>γ</text>
</annotation>
<annotation id="79">
<infon key="score">0.9991721</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5060" length="1"/>
<text>ζ</text>
</annotation>
<annotation id="80">
<infon key="score">0.99910283</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5063" length="1"/>
<text>δ</text>
</annotation>
<annotation id="81">
<infon key="score">0.9991536</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:00:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5069" length="1"/>
<text>ε</text>
</annotation>
<annotation id="678">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:59:23Z</infon>
<location offset="5073" length="33"/>
<text>N6-adenine and N4-cytosine MTases</text>
</annotation>
<annotation id="82">
<infon key="score">0.99840933</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5224" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="83">
<infon key="score">0.9991761</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5234" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="84">
<infon key="score">0.999046</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5352" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="827">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:36:17Z</infon>
<location offset="5370" length="16"/>
<text>catalytic domain</text>
</annotation>
<annotation id="829">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:36:35Z</infon>
<location offset="5420" length="17"/>
<text>C-terminal domain</text>
</annotation>
<annotation id="711">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:49Z</infon>
<location offset="5441" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="713">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<location offset="5469" length="8"/>
<text>M.MboIIA</text>
</annotation>
<annotation id="716">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<location offset="5482" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="831">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:36:51Z</infon>
<location offset="5494" length="12"/>
<text>helix bundle</text>
</annotation>
<annotation id="85">
<infon key="score">0.99908864</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:02:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5510" length="6"/>
<text>EcoDam</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>5553</offset>
<text>DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development.</text>
<annotation id="671">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:29Z</infon>
<location offset="5553" length="15"/>
<text>DNA methylation</text>
</annotation>
<annotation id="86">
<infon key="score">0.9987413</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5593" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="87">
<infon key="score">0.99834085</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:37:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5614" length="29"/>
<text>DNA adenine methyltransferase</text>
</annotation>
<annotation id="88">
<infon key="score">0.9982779</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:03:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5722" length="22"/>
<text>Salmonella typhimurium</text>
</annotation>
<annotation id="89">
<infon key="score">0.9983058</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:03:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5749" length="20"/>
<text>Aeromonas hydrophila</text>
</annotation>
<annotation id="680">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:59Z</infon>
<location offset="5784" length="23"/>
<text>DNA adenine methylation</text>
</annotation>
<annotation id="693">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:16:42Z</infon>
<location offset="5884" length="23"/>
<text>DNA adenine methylation</text>
</annotation>
<annotation id="90">
<infon key="score">0.9145081</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:16:58Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5957" length="3"/>
<text>Dam</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>6031</offset>
<text>Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.</text>
<annotation id="91">
<infon key="score">0.9981073</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:48:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6031" length="19"/>
<text>Helicobacter pylori</text>
</annotation>
<annotation id="92">
<infon key="score">0.9708807</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:03:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6056" length="23"/>
<text>Gram-negative bacterium</text>
</annotation>
<annotation id="93">
<infon key="score">0.9986656</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6111" length="5"/>
<text>human</text>
</annotation>
<annotation id="94">
<infon key="score">0.9977002</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6253" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="95">
<infon key="score">0.9980526</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6426" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="96">
<infon key="score">0.9977798</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:48:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="6481" length="8"/>
<text>bacteria</text>
</annotation>
<annotation id="97">
<infon key="score">0.9855281</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6524" length="15"/>
<text>H. pylori 26695</text>
</annotation>
<annotation id="98">
<infon key="score">0.99871564</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:24:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6574" length="18"/>
<text>Methyltransferases</text>
</annotation>
<annotation id="99">
<infon key="score">0.99808294</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6626" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="721">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:46:13Z</infon>
<location offset="6651" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="100">
<infon key="score">0.9983508</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:24:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6666" length="17"/>
<text>DNA adenine MTase</text>
</annotation>
<annotation id="101">
<infon key="score">0.9968161</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6749" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="699">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="6766" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="720">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:45Z</infon>
<location offset="6780" length="9"/>
<text>M2.HpyAVI</text>
</annotation>
<annotation id="102">
<infon key="score">0.8533181</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6806" length="18"/>
<text>restriction enzyme</text>
</annotation>
<annotation id="700">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="6826" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="103">
<infon key="score">0.995255</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:36Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="6851" length="6"/>
<text>hp0050</text>
</annotation>
<annotation id="104">
<infon key="score">0.9986347</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6864" length="28"/>
<text>N6-adenine methyltransferase</text>
</annotation>
<annotation id="105">
<infon key="score">0.998701</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="6910" length="13"/>
<text>β-class MTase</text>
</annotation>
<annotation id="106">
<infon key="score">0.97984064</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:36Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="6999" length="11"/>
<text>5′-GAGG-3′,</text>
</annotation>
<annotation id="107">
<infon key="score">0.9948631</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:34Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7011" length="10"/>
<text>5′-GGAG-3′</text>
</annotation>
<annotation id="108">
<infon key="score">0.997119</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7025" length="10"/>
<text>5′-GAAG-3′</text>
</annotation>
<annotation id="109">
<infon key="score">0.65228903</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7051" length="8"/>
<text>adenines</text>
</annotation>
<annotation id="816">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:49Z</infon>
<location offset="7091" length="11"/>
<text>methylation</text>
</annotation>
<annotation id="110">
<infon key="score">0.64126253</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="7119" length="8"/>
<text>adenines</text>
</annotation>
<annotation id="111">
<infon key="score">0.9987367</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7182" length="17"/>
<text>N6-adenine MTases</text>
</annotation>
<annotation id="817">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:49Z</infon>
<location offset="7205" length="11"/>
<text>methylation</text>
</annotation>
<annotation id="112">
<infon key="score">0.99674714</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:10Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7229" length="10"/>
<text>5′-GAAG-3′</text>
</annotation>
<annotation id="701">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="7272" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="113">
<infon key="score">0.9982695</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="7332" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="114">
<infon key="score">0.9978259</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:08Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="7373" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="115">
<infon key="score">0.8282734</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7529" length="9"/>
<text>structure</text>
</annotation>
<annotation id="116">
<infon key="score">0.9972285</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:28:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="7574" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="702">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="7595" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="117">
<infon key="score">0.9966917</infon>
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<text>Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.</text>
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<text>The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1.</text>
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<text>Data collection and structure refinement statistics of M1.HpyAVI</text>
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<table frame="box" rules="all"><thead><tr><th align="left" valign="middle" rowspan="1" colspan="1"/><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI</th><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI-AdoMet complex</th></tr></thead><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Data collection</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Wavelength (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.0000</td><td align="left" valign="middle" rowspan="1" colspan="1">0.97772</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Space group</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>4<sub>3</sub>2<sub>1</sub>2</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>6<sub>5</sub></td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unit-cell parameters (Å, ˚)</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a</italic> = <italic>b</italic> = 69.73, <italic>c</italic> = 532.75<break/><italic>α = β = γ</italic> = 90</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a = b</italic> = 135.60, <italic>c</italic> = 265.15<break/><italic>α = β</italic> = 90, <italic>γ</italic> = 120</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Resolution range (Å) <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">49.09-3.00 (3.09-3.00)</td><td align="left" valign="middle" rowspan="1" colspan="1">48.91-3.10 (3.18-3.10)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unique reflections <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">27243</td><td align="left" valign="middle" rowspan="1" colspan="1">49833</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Multiplicity <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">3.7 (3.8)</td><td align="left" valign="middle" rowspan="1" colspan="1">5.6 (4.0)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Completeness (%)<xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">98.7 (98.9)</td><td align="left" valign="middle" rowspan="1" colspan="1">99.7 (97.8)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Mean <italic>I/δ</italic> (<italic>I</italic>) <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">12.1 (3.4)</td><td align="left" valign="middle" rowspan="1" colspan="1">14.0 (1.9)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Solvent content (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">58.67</td><td align="left" valign="middle" rowspan="1" colspan="1">61.96</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub>
<xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">0.073 (0.378)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.106 (0.769)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Structure refinement</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>work</sub></td><td align="left" valign="middle" rowspan="1" colspan="1">0.251</td><td align="left" valign="middle" rowspan="1" colspan="1">0.221</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>free</sub></td><td align="left" valign="middle" rowspan="1" colspan="1">0.308</td><td align="left" valign="middle" rowspan="1" colspan="1">0.276</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond lengths (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond angles (˚)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.408</td><td align="left" valign="middle" rowspan="1" colspan="1">1.651</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Ramachandran plot</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Favoured region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">89.44</td><td align="left" valign="middle" rowspan="1" colspan="1">91.44</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Allowed region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">9.58</td><td align="left" valign="middle" rowspan="1" colspan="1">7.11</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Outliers (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.99</td><td align="left" valign="middle" rowspan="1" colspan="1">1.45</td></tr></tbody></table>
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<text> M1.HpyAVI M1.HpyAVI-AdoMet complex Data collection Wavelength (Å) 1.0000 0.97772 Space group P43212 P65 Unit-cell parameters (Å, ˚) a = b = 69.73, c = 532.75α = β = γ = 90 a = b = 135.60, c = 265.15α = β = 90, γ = 120 Resolution range (Å) a 49.09-3.00 (3.09-3.00) 48.91-3.10 (3.18-3.10) Unique reflections a 27243 49833 Multiplicity a 3.7 (3.8) 5.6 (4.0) Completeness (%)a 98.7 (98.9) 99.7 (97.8) Mean I/δ (I) a 12.1 (3.4) 14.0 (1.9) Solvent content (%) 58.67 61.96 Rmergea 0.073 (0.378) 0.106 (0.769) Structure refinement Rwork 0.251 0.221 Rfree 0.308 0.276 R.m.s.d., bond lengths (Å) 0.007 0.007 R.m.s.d., bond angles (˚) 1.408 1.651 Ramachandran plot Favoured region (%) 89.44 91.44 Allowed region (%) 9.58 7.11 Outliers (%) 0.99 1.45 </text>
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<text>Values in parentheses are statistics of the highest resolution shell.</text>
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<text>Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1.</text>
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<infon key="updated_at">2023-09-15T15:10:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9974" length="5"/>
<text>32-61</text>
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<annotation id="147">
<infon key="score">0.9974658</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:10:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="9984" length="7"/>
<text>152-172</text>
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<annotation id="148">
<infon key="score">0.997647</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:10:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10001" length="10"/>
<text>structures</text>
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<annotation id="149">
<infon key="score">0.9979949</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:10:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10036" length="16"/>
<text>electron density</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10156</offset>
<text>The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.</text>
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<infon key="updated_at">2023-09-18T09:28:38Z</infon>
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<location offset="10164" length="10"/>
<text>structures</text>
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<annotation id="151">
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<infon key="updated_at">2023-09-18T08:20:46Z</infon>
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<location offset="10252" length="4"/>
<text>RMSD</text>
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<annotation id="706">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="10312" length="9"/>
<text>M1.HpyAVI</text>
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<annotation id="152">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:28:41Z</infon>
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<location offset="10340" length="10"/>
<text>structures</text>
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<annotation id="153">
<infon key="score">0.9984342</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10365" length="22"/>
<text>AdoMet-dependent MTase</text>
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<annotation id="760">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:14Z</infon>
<location offset="10427" length="7"/>
<text>β-sheet</text>
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<annotation id="763">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:33Z</infon>
<location offset="10450" length="9"/>
<text>α-helices</text>
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<annotation id="154">
<infon key="score">0.9966743</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:28:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10505" length="10"/>
<text>structures</text>
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<annotation id="155">
<infon key="score">0.99891853</infon>
<infon key="type">protein_type</infon>
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<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10540" length="6"/>
<text>MTases</text>
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<annotation id="811">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:30:04Z</infon>
<location offset="10554" length="7"/>
<text>helices</text>
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<annotation id="758">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:59Z</infon>
<location offset="10563" length="2"/>
<text>αA</text>
</annotation>
<annotation id="768">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:44Z</infon>
<location offset="10567" length="2"/>
<text>αB</text>
</annotation>
<annotation id="770">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:55Z</infon>
<location offset="10574" length="2"/>
<text>αZ</text>
</annotation>
<annotation id="761">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:14Z</infon>
<location offset="10617" length="7"/>
<text>β-sheet</text>
</annotation>
<annotation id="753">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:28Z</infon>
<location offset="10648" length="2"/>
<text>αD</text>
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<annotation id="773">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:06Z</infon>
<location offset="10652" length="2"/>
<text>αE</text>
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<annotation id="775">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:17Z</infon>
<location offset="10659" length="2"/>
<text>αC</text>
</annotation>
<annotation id="156">
<infon key="score">0.99812</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:25:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10695" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="783">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:27Z</infon>
<location offset="10738" length="17"/>
<text>α/β Rossmann fold</text>
</annotation>
<annotation id="809">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:29:40Z</infon>
<location offset="10761" length="15"/>
<text>catalytic motif</text>
</annotation>
<annotation id="780">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:09Z</infon>
<location offset="10777" length="4"/>
<text>DPPY</text>
</annotation>
<annotation id="788">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="10792" length="4"/>
<text>loop</text>
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<annotation id="754">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:28Z</infon>
<location offset="10808" length="2"/>
<text>αD</text>
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<annotation id="750">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:17Z</infon>
<location offset="10815" length="2"/>
<text>β4</text>
</annotation>
<annotation id="157">
<infon key="score">0.99921036</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10836" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="158">
<infon key="score">0.96160835</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:16Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10866" length="6"/>
<text>cavity</text>
</annotation>
<annotation id="789">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="10878" length="4"/>
<text>loop</text>
</annotation>
<annotation id="159">
<infon key="score">0.99757195</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:26:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10893" length="7"/>
<text>136-166</text>
</annotation>
<annotation id="801">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:43Z</infon>
<location offset="10918" length="2"/>
<text>β7</text>
</annotation>
<annotation id="771">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:55Z</infon>
<location offset="10925" length="2"/>
<text>αZ</text>
</annotation>
<annotation id="160">
<infon key="score">0.9987655</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10945" length="14"/>
<text>highly diverse</text>
</annotation>
<annotation id="161">
<infon key="score">0.9991721</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10976" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="695">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:28:47Z</infon>
<location offset="11010" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="806">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:29:16Z</infon>
<location offset="11031" length="12"/>
<text>hairpin loop</text>
</annotation>
<annotation id="162">
<infon key="score">0.9974844</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:26:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11054" length="7"/>
<text>101-133</text>
</annotation>
<annotation id="804">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:56Z</infon>
<location offset="11072" length="2"/>
<text>β6</text>
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<annotation id="802">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:43Z</infon>
<location offset="11079" length="2"/>
<text>β7</text>
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<annotation id="163">
<infon key="score">0.9946524</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11109" length="3"/>
<text>DNA</text>
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<annotation id="784">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:47Z</infon>
<location offset="11120" length="12"/>
<text>minor groove</text>
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<annotation id="714">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<location offset="11187" length="8"/>
<text>M.MboIIA</text>
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<annotation id="717">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<location offset="11197" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="722">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:47:39Z</infon>
<location offset="11208" length="7"/>
<text>M.pvuII</text>
</annotation>
<annotation id="164">
<infon key="score">0.7994238</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11221" length="7"/>
<text>missing</text>
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<annotation id="790">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="11229" length="4"/>
<text>loop</text>
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<annotation id="165">
<infon key="score">0.99738026</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:26:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11244" length="5"/>
<text>33-58</text>
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<annotation id="166">
<infon key="score">0.9981224</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:28:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11258" length="9"/>
<text>structure</text>
</annotation>
<annotation id="707">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="11271" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="800">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:26Z</infon>
<location offset="11296" length="6"/>
<text>loop I</text>
</annotation>
<annotation id="712">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:50Z</infon>
<location offset="11306" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="167">
<infon key="score">0.99823296</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:29:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11344" length="9"/>
<text>structure</text>
</annotation>
<annotation id="694">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:26:42Z</infon>
<location offset="11354" length="11"/>
<text>without DNA</text>
</annotation>
<annotation id="785">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="11372" length="4"/>
<text>loop</text>
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<annotation id="880">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:29:06Z</infon>
<location offset="11391" length="12"/>
<text>well ordered</text>
</annotation>
<annotation id="723">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:48:16Z</infon>
<location offset="11410" length="28"/>
<text>M.TaqI-DNA complex structure</text>
</annotation>
<annotation id="672">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:29Z</infon>
<location offset="11479" length="15"/>
<text>DNA methylation</text>
</annotation>
<annotation id="168">
<infon key="score">0.65298414</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:22Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11522" length="7"/>
<text>adenine</text>
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<annotation id="696">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:28:58Z</infon>
<location offset="11555" length="3"/>
<text>DNA</text>
</annotation>
</passage>
<passage>
<infon key="file">oncotarget-07-40965-g001.jpg</infon>
<infon key="id">F1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>11634</offset>
<text>Overall structure of M1.HpyAVI</text>
<annotation id="169">
<infon key="score">0.9969964</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:29:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11642" length="9"/>
<text>structure</text>
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<annotation id="708">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="11655" length="9"/>
<text>M1.HpyAVI</text>
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</passage>
<passage>
<infon key="file">oncotarget-07-40965-g001.jpg</infon>
<infon key="id">F1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>11665</offset>
<text>A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green.</text>
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<text>Free</text>
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<annotation id="171">
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<infon key="updated_at">2023-09-15T14:49:26Z</infon>
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<text>AdoMet-bound</text>
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<annotation id="709">
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<infon key="updated_at">2023-09-15T15:44:17Z</infon>
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<text>M1.HpyAVI</text>
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<annotation id="172">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:44Z</infon>
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<location offset="11742" length="22"/>
<text>AdoMet-dependent MTase</text>
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<annotation id="759">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<text>β-sheet</text>
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<annotation id="762">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:33Z</infon>
<location offset="11818" length="9"/>
<text>α-helices</text>
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<annotation id="757">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:59Z</infon>
<location offset="11829" length="2"/>
<text>αA</text>
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<annotation id="767">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:43Z</infon>
<location offset="11833" length="2"/>
<text>αB</text>
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<annotation id="769">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:54Z</infon>
<location offset="11837" length="2"/>
<text>αZ</text>
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<annotation id="755">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:28Z</infon>
<location offset="11856" length="2"/>
<text>αD</text>
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<annotation id="772">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:06Z</infon>
<location offset="11860" length="2"/>
<text>αE</text>
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<annotation id="774">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:16Z</infon>
<location offset="11864" length="2"/>
<text>αC</text>
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<annotation id="173">
<infon key="score">0.9992446</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11899" length="6"/>
<text>AdoMet</text>
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<annotation id="174">
<infon key="score">0.99239594</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:31:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11909" length="8"/>
<text>bound in</text>
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<annotation id="175">
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:31:30Z</infon>
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<location offset="11920" length="6"/>
<text>cavity</text>
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<annotation id="176">
<infon key="score">0.9990746</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:31:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11936" length="9"/>
<text>conserved</text>
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<annotation id="779">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:08Z</infon>
<location offset="11968" length="4"/>
<text>DPPY</text>
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<annotation id="764">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:33Z</infon>
<location offset="12096" length="9"/>
<text>α-helices</text>
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<annotation id="777">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:57Z</infon>
<location offset="12110" length="9"/>
<text>β-strands</text>
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<annotation id="177">
<infon key="score">0.99922407</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12201" length="6"/>
<text>MTases</text>
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<annotation id="178">
<infon key="score">0.9991905</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12213" length="6"/>
<text>AdoMet</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>12286</offset>
<text>Dimeric state of M1.HpyAVI in crystal and solution</text>
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<infon key="score">0.9984648</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12286" length="7"/>
<text>Dimeric</text>
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<annotation id="710">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:44:17Z</infon>
<location offset="12303" length="9"/>
<text>M1.HpyAVI</text>
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<annotation id="180">
<infon key="score">0.9944754</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:48:43Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>crystal</text>
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<annotation id="181">
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:03:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12328" length="8"/>
<text>solution</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>12337</offset>
<text>Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.</text>
<annotation id="182">
<infon key="score">0.99734926</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12371" length="10"/>
<text>DNA MTases</text>
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<annotation id="724">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:01Z</infon>
<location offset="12388" length="7"/>
<text>M.BamHI</text>
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<annotation id="725">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:16Z</infon>
<location offset="12400" length="7"/>
<text>M.EcoRI</text>
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<annotation id="183">
<infon key="score">0.99874026</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12418" length="7"/>
<text>monomer</text>
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<annotation id="184">
<infon key="score">0.82037216</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12473" length="3"/>
<text>DNA</text>
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<annotation id="185">
<infon key="score">0.9990522</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:47Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12501" length="5"/>
<text>MTase</text>
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<annotation id="186">
<infon key="score">0.9989624</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:41:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12510" length="14"/>
<text>hemimethylated</text>
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<annotation id="818">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:49Z</infon>
<location offset="12559" length="11"/>
<text>methylation</text>
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<annotation id="187">
<infon key="score">0.99889743</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:41:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12598" length="16"/>
<text>fully methylated</text>
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<annotation id="188">
<infon key="score">0.9988433</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12643" length="7"/>
<text>dimeric</text>
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<annotation id="189">
<infon key="score">0.9972397</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12651" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="190">
<infon key="score">0.9688514</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:40Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12726" length="7"/>
<text>M.DpnII</text>
</annotation>
<annotation id="191">
<infon key="score">0.90760726</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12735" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="192">
<infon key="score">0.94044906</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:49Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12743" length="6"/>
<text>M.KpnI</text>
</annotation>
<annotation id="193">
<infon key="score">0.99051577</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12755" length="8"/>
<text>M.MboIIA</text>
</annotation>
<annotation id="194">
<infon key="score">0.9987752</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12783" length="6"/>
<text>dimers</text>
</annotation>
<annotation id="195">
<infon key="score">0.9991013</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12824" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="196">
<infon key="score">0.98035747</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12841" length="8"/>
<text>M.MboIIA</text>
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<annotation id="197">
<infon key="score">0.88078374</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12851" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="198">
<infon key="score">0.9984231</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:51Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12862" length="7"/>
<text>TTH0409</text>
</annotation>
<annotation id="199">
<infon key="score">0.998779</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12894" length="6"/>
<text>dimers</text>
</annotation>
<annotation id="200">
<infon key="score">0.9989133</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12904" length="18"/>
<text>crystal structures</text>
</annotation>
<annotation id="201">
<infon key="score">0.9965406</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12942" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="202">
<infon key="score">0.9439273</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12961" length="7"/>
<text>M.CcrMI</text>
</annotation>
<annotation id="203">
<infon key="score">0.99796015</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12977" length="26"/>
<text>Bacillus amyloliquefaciens</text>
</annotation>
<annotation id="204">
<infon key="score">0.9990553</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13004" length="5"/>
<text>MTase</text>
</annotation>
<annotation id="205">
<infon key="score">0.9988293</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13026" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="206">
<infon key="score">0.9987846</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13037" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="207">
<infon key="score">0.854749</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:57Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13050" length="3"/>
<text>DNA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13063</offset>
<text>According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).</text>
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<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<location offset="13105" length="9"/>
<text>conserved</text>
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<infon key="score">0.6177626</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="13124" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="726">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:50:56Z</infon>
<location offset="13149" length="10"/>
<text>β-subgroup</text>
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<annotation id="209">
<infon key="score">0.99775726</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13172" length="9"/>
<text>conserved</text>
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<annotation id="776">
<infon key="type">structure_element</infon>
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<text>A. A conserved interface area of β-class MTases is defined in M1.HpyAVI. Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta. D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution. FPLC system coupled to a Superdex 75 10/300 column. Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.</text>
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<text>To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).</text>
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<text>Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state.</text>
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<infon key="updated_at">2023-09-18T08:43:31Z</infon>
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<location offset="15205" length="9"/>
<text>monomeric</text>
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<annotation id="263">
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<infon key="updated_at">2023-09-18T09:37:03Z</infon>
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<location offset="15289" length="8"/>
<text>arginine</text>
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<infon key="annotator">cleaner0</infon>
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<text>arginine</text>
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<text>glycerol</text>
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<passage>
<infon key="section_type">RESULTS</infon>
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<text>Structure comparisons</text>
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<location offset="15513" length="21"/>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15535</offset>
<text>As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.</text>
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<infon key="updated_at">2023-09-18T09:26:08Z</infon>
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<text>M.MboIIA</text>
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<infon key="updated_at">2023-09-15T15:45:22Z</infon>
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<text>M.RsrI</text>
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<infon key="updated_at">2023-09-18T08:45:12Z</infon>
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<location offset="15714" length="15"/>
<text>Superimposition</text>
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<infon key="updated_at">2023-09-15T14:49:13Z</infon>
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<location offset="15733" length="9"/>
<text>M1.HpyAVI</text>
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<infon key="updated_at">2023-09-18T08:45:56Z</infon>
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<location offset="15758" length="5"/>
<text>RMSDs</text>
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<text>TRD</text>
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<infon key="updated_at">2023-09-18T10:05:30Z</infon>
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<location offset="15911" length="7"/>
<text>133-163</text>
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<location offset="15922" length="9"/>
<text>M1.HpyAVI</text>
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<infon key="updated_at">2023-09-18T08:25:33Z</infon>
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<text>α-helices</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:26:58Z</infon>
<location offset="16115" length="9"/>
<text>β-strands</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
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<text>TRD</text>
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<infon key="type">protein</infon>
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<infon key="updated_at">2023-09-15T14:49:13Z</infon>
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<location offset="16137" length="9"/>
<text>M1.HpyAVI</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:00Z</infon>
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<text>lacking</text>
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<text>TRD</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:11Z</infon>
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<text>DNA</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:44:36Z</infon>
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<text>major groove</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16374" length="3"/>
<text>DNA</text>
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<infon key="score">0.9986234</infon>
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<infon key="updated_at">2023-09-18T09:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16446" length="15"/>
<text>highly flexible</text>
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<annotation id="791">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="16462" length="4"/>
<text>loop</text>
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<annotation id="749">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:17Z</infon>
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<text>β4</text>
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<annotation id="752">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-18T08:24:28Z</infon>
<location offset="16482" length="2"/>
<text>αD</text>
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<infon key="updated_at">2023-09-18T10:05:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16495" length="5"/>
<text>33-58</text>
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<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16505" length="9"/>
<text>M1.HpyAVI</text>
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:29:48Z</infon>
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<location offset="16543" length="10"/>
<text>structures</text>
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<infon key="updated_at">2023-09-18T09:29:51Z</infon>
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<location offset="16573" length="10"/>
<text>structures</text>
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<infon key="type">protein</infon>
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<text>M.MboIIA</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
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<text>M.RsrI</text>
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<infon key="updated_at">2023-09-18T08:45:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16608" length="18"/>
<text>Sequence alignment</text>
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<infon key="score">0.9674974</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16656" length="9"/>
<text>M1.HpyAVI</text>
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<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16763" length="9"/>
<text>H. pylori</text>
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<infon key="type">protein_state</infon>
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<infon key="updated_at">2023-09-18T09:31:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16785" length="8"/>
<text>flexible</text>
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<passage>
<infon key="file">oncotarget-07-40965-g003.jpg</infon>
<infon key="id">F3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>16816</offset>
<text>Structural comparisons between M1.HpyAVI and other DNA MTases</text>
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<location offset="16816" length="22"/>
<text>Structural comparisons</text>
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<text>M1.HpyAVI</text>
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<infon key="updated_at">2023-09-15T14:51:23Z</infon>
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<text>DNA MTases</text>
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<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>16878</offset>
<text>A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.</text>
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<text>M1.HpyAVI</text>
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<infon key="updated_at">2023-09-18T08:21:39Z</infon>
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<infon key="identifier">PR:</infon>
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<text>M.TaqI</text>
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<infon key="type">protein</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="16949" length="9"/>
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<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:32:38Z</infon>
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<infon key="updated_at">2023-09-18T08:23:59Z</infon>
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<text>TRD</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:31:23Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>structure-rich</text>
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<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-18T08:23:59Z</infon>
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<text>TRD</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17042" length="8"/>
<text>M.MboIIA</text>
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<annotation id="719">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
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<text>M.RsrI</text>
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<infon key="score">0.99877506</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17063" length="8"/>
<text>TTHA0409</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:46:25Z</infon>
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<text>DNA-binding domain</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17108" length="4"/>
<text>DpnM</text>
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<annotation id="299">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17117" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="739">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="17156" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="646">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="17163" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="300">
<infon key="score">0.97637516</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17174" length="8"/>
<text>M.MboIIA</text>
</annotation>
<annotation id="301">
<infon key="score">0.84092015</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17184" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="302">
<infon key="score">0.9985129</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17195" length="8"/>
<text>TTHA0409</text>
</annotation>
<annotation id="751">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:17Z</infon>
<location offset="17234" length="2"/>
<text>β4</text>
</annotation>
<annotation id="756">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:24:28Z</infon>
<location offset="17241" length="2"/>
<text>αD</text>
</annotation>
<annotation id="303">
<infon key="score">0.99199146</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17247" length="8"/>
<text>M.MboIIA</text>
</annotation>
<annotation id="304">
<infon key="score">0.98690724</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17260" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="835">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:46:25Z</infon>
<location offset="17280" length="18"/>
<text>DNA-binding domain</text>
</annotation>
<annotation id="305">
<infon key="score">0.9993131</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17302" length="4"/>
<text>DpnM</text>
</annotation>
<annotation id="830">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:36:35Z</infon>
<location offset="17326" length="17"/>
<text>C-terminal domain</text>
</annotation>
<annotation id="306">
<infon key="score">0.99063236</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17347" length="6"/>
<text>M.TaqI</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17375</offset>
<text>N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).</text>
<annotation id="730">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:01Z</infon>
<location offset="17375" length="33"/>
<text>N6-adenine and N4-cytosine MTases</text>
</annotation>
<annotation id="307">
<infon key="score">0.9988351</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:03:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17484" length="21"/>
<text>Structural comparison</text>
</annotation>
<annotation id="308">
<infon key="score">0.9828641</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17514" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="309">
<infon key="score">0.99869984</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:20:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17539" length="25"/>
<text>β-class N4 cytosine MTase</text>
</annotation>
<annotation id="310">
<infon key="score">0.9993332</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:04Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17571" length="8"/>
<text>TTHA0409</text>
</annotation>
<annotation id="311">
<infon key="score">0.998705</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:20:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17638" length="4"/>
<text>RMSD</text>
</annotation>
<annotation id="740">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="17763" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="312">
<infon key="score">0.9986004</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:20:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17778" length="10"/>
<text>structures</text>
</annotation>
<annotation id="766">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:25:33Z</infon>
<location offset="17829" length="9"/>
<text>α-helices</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>17863</offset>
<text>M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.</text>
<annotation id="313">
<infon key="score">0.71178216</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17863" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="314">
<infon key="score">0.9987031</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17942" length="17"/>
<text>N6-adenine MTases</text>
</annotation>
<annotation id="727">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:34Z</infon>
<location offset="17995" length="7"/>
<text>α-class</text>
</annotation>
<annotation id="315">
<infon key="score">0.99924123</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18003" length="4"/>
<text>DpnM</text>
</annotation>
<annotation id="728">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:58Z</infon>
<location offset="18030" length="7"/>
<text>γ-class</text>
</annotation>
<annotation id="316">
<infon key="score">0.992579</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18038" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="317">
<infon key="score">0.9981804</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:45:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18082" length="5"/>
<text>RMSDs</text>
</annotation>
<annotation id="318">
<infon key="score">0.9785749</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:32:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18138" length="4"/>
<text>lack</text>
</annotation>
<annotation id="729">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:22:41Z</infon>
<location offset="18145" length="16"/>
<text>counterpart loop</text>
</annotation>
<annotation id="741">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="18177" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="319">
<infon key="score">0.9628337</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18184" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="320">
<infon key="score">0.60606277</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18235" length="3"/>
<text>DNA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18281</offset>
<text>Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.</text>
<annotation id="321">
<infon key="score">0.9905677</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18295" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="322">
<infon key="score">0.8527761</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:32:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18317" length="15"/>
<text>long disordered</text>
</annotation>
<annotation id="742">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="18333" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="323">
<infon key="score">0.84751034</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:32:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18372" length="24"/>
<text>secondary structure-rich</text>
</annotation>
<annotation id="743">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="18397" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="731">
<infon key="type">protein_type</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:19Z</infon>
<location offset="18410" length="40"/>
<text>β-class N6 adenine or N4 cytosine MTases</text>
</annotation>
<annotation id="324">
<infon key="score">0.99855304</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18494" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="325">
<infon key="score">0.99791336</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18625" length="9"/>
<text>H. pylori</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>18646</offset>
<text>AdoMet-binding pocket</text>
<annotation id="326">
<infon key="score">0.99897456</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:47:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18646" length="21"/>
<text>AdoMet-binding pocket</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18668</offset>
<text>The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.</text>
<annotation id="327">
<infon key="score">0.9991107</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:26Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18672" length="23"/>
<text>cofactor binding pocket</text>
</annotation>
<annotation id="328">
<infon key="score">0.9654598</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18699" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="329">
<infon key="score">0.9948056</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18735" length="3"/>
<text>7-9</text>
</annotation>
<annotation id="330">
<infon key="score">0.9951399</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18740" length="5"/>
<text>29-31</text>
</annotation>
<annotation id="331">
<infon key="score">0.9965372</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18747" length="7"/>
<text>165-167</text>
</annotation>
<annotation id="332">
<infon key="score">0.99617857</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18756" length="7"/>
<text>216-218</text>
</annotation>
<annotation id="333">
<infon key="score">0.9353764</infon>
<infon key="type">residue_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:07:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18768" length="3"/>
<text>221</text>
</annotation>
<annotation id="334">
<infon key="score">0.99897015</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18795" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="335">
<infon key="score">0.9982679</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18819" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="336">
<infon key="score">0.9966004</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18833" length="13"/>
<text>hydrogen bond</text>
</annotation>
<annotation id="337">
<infon key="score">0.99955183</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18855" length="3"/>
<text>D29</text>
</annotation>
<annotation id="810">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:29:41Z</infon>
<location offset="18866" length="15"/>
<text>catalytic motif</text>
</annotation>
<annotation id="781">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:09Z</infon>
<location offset="18882" length="4"/>
<text>DPPY</text>
</annotation>
<annotation id="338">
<infon key="score">0.83229464</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:33:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18910" length="5"/>
<text>bound</text>
</annotation>
<annotation id="339">
<infon key="score">0.99907744</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="18916" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="340">
<infon key="score">0.99916065</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18945" length="5"/>
<text>MTase</text>
</annotation>
<annotation id="341">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18951" length="10"/>
<text>structures</text>
</annotation>
<annotation id="342">
<infon key="score">0.9995264</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18972" length="2"/>
<text>D8</text>
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<annotation id="343">
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<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18979" length="2"/>
<text>A9</text>
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<annotation id="344">
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<infon key="updated_at">2023-09-18T08:47:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="18987" length="14"/>
<text>hydrogen-bonds</text>
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<annotation id="345">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19024" length="6"/>
<text>purine</text>
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<annotation id="346">
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<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19055" length="4"/>
<text>E216</text>
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<annotation id="347">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:47:51Z</infon>
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<location offset="19076" length="16"/>
<text>hydrogen bonding</text>
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<annotation id="348">
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<infon key="updated_at">2023-09-18T09:37:35Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19126" length="6"/>
<text>ribose</text>
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<annotation id="349">
<infon key="score">0.9994992</infon>
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<infon key="updated_at">2023-09-18T08:48:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19147" length="4"/>
<text>H168</text>
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<annotation id="350">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19153" length="4"/>
<text>T200</text>
</annotation>
<annotation id="351">
<infon key="score">0.9995173</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19162" length="4"/>
<text>S198</text>
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<annotation id="352">
<infon key="score">0.99901843</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19200" length="6"/>
<text>AdoMet</text>
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<annotation id="353">
<infon key="score">0.99861515</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19208" length="13"/>
<text>Superposition</text>
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<annotation id="354">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19225" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="355">
<infon key="score">0.9965333</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19249" length="10"/>
<text>structures</text>
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<annotation id="356">
<infon key="score">0.53536797</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19322" length="16"/>
<text>rather conserved</text>
</annotation>
<annotation id="357">
<infon key="score">0.9970794</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19350" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="878">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:54Z</infon>
<location offset="19404" length="5"/>
<text>bound</text>
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<annotation id="358">
<infon key="score">0.9969217</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19431" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="359">
<infon key="score">0.9959515</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:26:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19467" length="10"/>
<text>absence of</text>
</annotation>
<annotation id="360">
<infon key="score">0.99926144</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19508" length="9"/>
<text>conserved</text>
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<annotation id="675">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<location offset="19518" length="6"/>
<text>AdoMet</text>
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<annotation id="836">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:19Z</infon>
<location offset="19539" length="5"/>
<text>FXGXG</text>
</annotation>
<annotation id="361">
<infon key="score">0.9978817</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:29:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19553" length="9"/>
<text>structure</text>
</annotation>
</passage>
<passage>
<infon key="file">oncotarget-07-40965-g004.jpg</infon>
<infon key="id">F4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>19568</offset>
<text>Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding</text>
<annotation id="362">
<infon key="score">0.9989014</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:03:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19568" length="35"/>
<text>Structural and biochemical analyses</text>
</annotation>
<annotation id="363">
<infon key="score">0.8775193</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19615" length="9"/>
<text>conserved</text>
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<annotation id="364">
<infon key="score">0.99950695</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19634" length="3"/>
<text>D29</text>
</annotation>
<annotation id="365">
<infon key="score">0.9995067</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19642" length="4"/>
<text>E216</text>
</annotation>
<annotation id="366">
<infon key="score">0.9987552</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19671" length="6"/>
<text>AdoMet</text>
</annotation>
</passage>
<passage>
<infon key="file">oncotarget-07-40965-g004.jpg</infon>
<infon key="id">F4</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>19686</offset>
<text>A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).</text>
<annotation id="367">
<infon key="score">0.9989493</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19693" length="23"/>
<text>cofactor-binding cavity</text>
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<annotation id="647">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="19720" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="368">
<infon key="score">0.99707735</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:41:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19766" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="369">
<infon key="score">0.99910104</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19786" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="370">
<infon key="score">0.9961052</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19832" length="13"/>
<text>hydrogen bond</text>
</annotation>
<annotation id="371">
<infon key="score">0.9977174</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19860" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="372">
<infon key="score">0.9991123</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19877" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="373">
<infon key="score">0.9963967</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19891" length="10"/>
<text>structures</text>
</annotation>
<annotation id="648">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="19905" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="374">
<infon key="score">0.99905735</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19924" length="4"/>
<text>DpnM</text>
</annotation>
<annotation id="375">
<infon key="score">0.9953942</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19942" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="376">
<infon key="score">0.99904925</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19963" length="6"/>
<text>AdoMet</text>
</annotation>
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<infon key="score">0.9956592</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="19991" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="378">
<infon key="score">0.99713755</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20033" length="25"/>
<text>Cofactor binding affinity</text>
</annotation>
<annotation id="379">
<infon key="score">0.99916446</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20062" length="2"/>
<text>wt</text>
</annotation>
<annotation id="380">
<infon key="score">0.9976549</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20066" length="7"/>
<text>mutants</text>
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<annotation id="649">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="20074" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="381">
<infon key="score">0.99877167</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20105" length="25"/>
<text>microscale thermophoresis</text>
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<infon key="score">0.998623</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20132" length="3"/>
<text>MST</text>
</annotation>
<annotation id="383">
<infon key="score">0.9986141</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20142" length="16"/>
<text>binding affinity</text>
</annotation>
<annotation id="650">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="20205" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="384">
<infon key="score">0.8690669</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:33:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20227" length="9"/>
<text>unlabeled</text>
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<annotation id="385">
<infon key="score">0.9990721</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20237" length="6"/>
<text>AdoMet</text>
</annotation>
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<infon key="score">0.9992286</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20322" length="6"/>
<text>AdoMet</text>
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<annotation id="387">
<infon key="score">0.9387604</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20349" length="8"/>
<text>titrated</text>
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<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="20388" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="388">
<infon key="score">0.99916697</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20398" length="2"/>
<text>wt</text>
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<infon key="score">0.99836916</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20401" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="390">
<infon key="score">0.99854326</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20431" length="21"/>
<text>dissociation constant</text>
</annotation>
<annotation id="391">
<infon key="score">0.99862623</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20454" length="2"/>
<text>KD</text>
</annotation>
<annotation id="392">
<infon key="score">0.9983845</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20514" length="8"/>
<text>isotherm</text>
</annotation>
<annotation id="652">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="20548" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="393">
<infon key="score">0.99194276</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20558" length="2"/>
<text>wt</text>
</annotation>
<annotation id="841">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:52:11Z</infon>
<location offset="20573" length="13"/>
<text>M1.HpyAVI-D8A</text>
</annotation>
<annotation id="842">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:52:32Z</infon>
<location offset="20601" length="14"/>
<text>M1.HpyAVI-D29A</text>
</annotation>
<annotation id="843">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:52:53Z</infon>
<location offset="20624" length="15"/>
<text>M1.HpyAVI-H168A</text>
</annotation>
<annotation id="839">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:51:07Z</infon>
<location offset="20655" length="15"/>
<text>M1.HpyAVI-S198A</text>
</annotation>
<annotation id="840">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:51:26Z</infon>
<location offset="20686" length="15"/>
<text>M1.HpyAVI-T200A</text>
</annotation>
<annotation id="844">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:53:13Z</infon>
<location offset="20717" length="15"/>
<text>M1.HpyAVI-E216A</text>
</annotation>
<annotation id="394">
<infon key="score">0.9817704</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:53:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20853" length="21"/>
<text>DNA methyltransferase</text>
</annotation>
<annotation id="395">
<infon key="score">0.9427799</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:33:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20887" length="9"/>
<text>wide type</text>
</annotation>
<annotation id="396">
<infon key="score">0.87695163</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20913" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="397">
<infon key="score">0.9975476</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:53:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20941" length="17"/>
<text>radioactive assay</text>
</annotation>
<annotation id="845">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:19Z</infon>
<location offset="20960" length="11"/>
<text>[3H]-methyl</text>
</annotation>
<annotation id="398">
<infon key="score">0.78140366</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:41Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20994" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="399">
<infon key="score">0.9070639</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:45Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21009" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="400">
<infon key="score">0.99856466</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21168" length="13"/>
<text>Superposition</text>
</annotation>
<annotation id="653">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="21185" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="401">
<infon key="score">0.99369144</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:05Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21208" length="8"/>
<text>M.MboIIA</text>
</annotation>
<annotation id="402">
<infon key="score">0.9863444</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:45:23Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21228" length="6"/>
<text>M.RsrI</text>
</annotation>
<annotation id="403">
<infon key="score">0.99943274</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21255" length="3"/>
<text>D29</text>
</annotation>
<annotation id="404">
<infon key="score">0.9994393</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21263" length="4"/>
<text>E216</text>
</annotation>
<annotation id="405">
<infon key="score">0.9114434</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21272" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="406">
<infon key="score">0.9983665</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21298" length="10"/>
<text>DNA MTases</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>21373</offset>
<text>To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.</text>
<annotation id="407">
<infon key="score">0.69778824</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:21Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21445" length="14"/>
<text>single mutants</text>
</annotation>
<annotation id="408">
<infon key="score">0.99535275</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:03:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21463" length="9"/>
<text>replacing</text>
</annotation>
<annotation id="409">
<infon key="score">0.99935883</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21473" length="2"/>
<text>D8</text>
</annotation>
<annotation id="410">
<infon key="score">0.9994481</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21477" length="3"/>
<text>D29</text>
</annotation>
<annotation id="411">
<infon key="score">0.9994443</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21482" length="4"/>
<text>H168</text>
</annotation>
<annotation id="412">
<infon key="score">0.99945134</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21488" length="4"/>
<text>S198</text>
</annotation>
<annotation id="413">
<infon key="score">0.9994473</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21494" length="4"/>
<text>T200</text>
</annotation>
<annotation id="414">
<infon key="score">0.99935776</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21500" length="4"/>
<text>E216</text>
</annotation>
<annotation id="415">
<infon key="score">0.9922931</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21510" length="7"/>
<text>alanine</text>
</annotation>
<annotation id="416">
<infon key="score">0.9984741</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21541" length="23"/>
<text>ligand binding affinity</text>
</annotation>
<annotation id="417">
<infon key="score">0.9987993</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21571" length="25"/>
<text>microscale thermophoresis</text>
</annotation>
<annotation id="418">
<infon key="score">0.9986884</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21598" length="3"/>
<text>MST</text>
</annotation>
<annotation id="419">
<infon key="score">0.99891734</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21652" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="420">
<infon key="score">0.8193448</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21675" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="421">
<infon key="score">0.9980161</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:54:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21715" length="2"/>
<text>KD</text>
</annotation>
<annotation id="422">
<infon key="score">0.9979571</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:40Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21740" length="4"/>
<text>D29A</text>
</annotation>
<annotation id="423">
<infon key="score">0.99816173</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21749" length="5"/>
<text>E216A</text>
</annotation>
<annotation id="424">
<infon key="score">0.95574015</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21755" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="425">
<infon key="score">0.8913477</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21776" length="23"/>
<text>protein-AdoMet affinity</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>21808</offset>
<text>The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.</text>
<annotation id="426">
<infon key="score">0.99543846</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:41:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21839" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="427">
<infon key="score">0.99947375</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21864" length="3"/>
<text>D29</text>
</annotation>
<annotation id="428">
<infon key="score">0.9994604</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21872" length="4"/>
<text>E216</text>
</annotation>
<annotation id="429">
<infon key="score">0.9984509</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="21882" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="430">
<infon key="score">0.99554175</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="21942" length="8"/>
<text>Mutation</text>
</annotation>
<annotation id="853">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="21996" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="654">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="22024" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="431">
<infon key="score">0.99947006</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22053" length="3"/>
<text>D29</text>
</annotation>
<annotation id="432">
<infon key="score">0.93916696</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22096" length="21"/>
<text>catalytic active site</text>
</annotation>
<annotation id="433">
<infon key="score">0.99586785</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22118" length="4"/>
<text>DPPY</text>
</annotation>
<annotation id="434">
<infon key="score">0.9994399</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22140" length="4"/>
<text>E216</text>
</annotation>
<annotation id="435">
<infon key="score">0.99761105</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22190" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="825">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:35:41Z</infon>
<location offset="22200" length="10"/>
<text>amino acid</text>
</annotation>
<annotation id="436">
<infon key="score">0.998909</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22222" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="437">
<infon key="score">0.9994332</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22242" length="4"/>
<text>E216</text>
</annotation>
<annotation id="846">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:11Z</infon>
<location offset="22270" length="2"/>
<text>β2</text>
</annotation>
<annotation id="438">
<infon key="score">0.51602983</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22314" length="6"/>
<text>ribose</text>
</annotation>
<annotation id="439">
<infon key="score">0.9988117</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22324" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="440">
<infon key="score">0.99830014</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:03:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22332" length="11"/>
<text>Replacement</text>
</annotation>
<annotation id="441">
<infon key="score">0.9955804</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22363" length="7"/>
<text>alanine</text>
</annotation>
<annotation id="442">
<infon key="score">0.9938001</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:41:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22400" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="443">
<infon key="score">0.9766973</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="22419" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="847">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="22462" length="6"/>
<text>methyl</text>
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<annotation id="885">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:04:00Z</infon>
<location offset="22512" length="29"/>
<text>[3H]AdoMet radiological assay</text>
</annotation>
<annotation id="854">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="22570" length="6"/>
<text>methyl</text>
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<annotation id="444">
<infon key="score">0.87604487</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22602" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="445">
<infon key="score">0.9986948</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:04:05Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22648" length="18"/>
<text>radiological assay</text>
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<annotation id="446">
<infon key="score">0.9986708</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22688" length="3"/>
<text>MST</text>
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<annotation id="447">
<infon key="score">0.998423</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:57:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22709" length="4"/>
<text>D29A</text>
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<annotation id="448">
<infon key="score">0.99846244</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:57:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22718" length="5"/>
<text>E216A</text>
</annotation>
<annotation id="449">
<infon key="score">0.98877406</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22724" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="855">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="22752" length="6"/>
<text>methyl</text>
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<annotation id="837">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<location offset="22790" length="7"/>
<text>mutants</text>
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<annotation id="450">
<infon key="score">0.6297416</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:31:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22816" length="17"/>
<text>methyltransferase</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>22847</offset>
<text>As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.</text>
<annotation id="863">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:23Z</infon>
<location offset="22872" length="5"/>
<text>FXGXG</text>
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<annotation id="451">
<infon key="score">0.99913895</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22883" length="9"/>
<text>conserved</text>
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<annotation id="676">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<location offset="22893" length="6"/>
<text>AdoMet</text>
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<annotation id="452">
<infon key="score">0.99815035</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22917" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="453">
<infon key="score">0.7042762</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22942" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="454">
<infon key="score">0.73182875</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:07:45Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22954" length="5"/>
<text>FMGSG</text>
</annotation>
<annotation id="455">
<infon key="score">0.9881989</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22964" length="7"/>
<text>alanine</text>
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<annotation id="826">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:35:41Z</infon>
<location offset="22982" length="10"/>
<text>amino acid</text>
</annotation>
<annotation id="456">
<infon key="score">0.9988029</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:07:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23013" length="5"/>
<text>F195A</text>
</annotation>
<annotation id="457">
<infon key="score">0.99854386</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23019" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="458">
<infon key="score">0.99794036</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23145" length="23"/>
<text>ligand binding affinity</text>
</annotation>
<annotation id="856">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="23173" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="838">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<location offset="23211" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="459">
<infon key="score">0.9985948</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:50:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23225" length="3"/>
<text>MST</text>
</annotation>
<annotation id="460">
<infon key="score">0.91924936</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:04:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23235" length="18"/>
<text>radiological assay</text>
</annotation>
<annotation id="461">
<infon key="score">0.99950063</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23269" length="4"/>
<text>G197</text>
</annotation>
<annotation id="462">
<infon key="score">0.9926398</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23299" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="463">
<infon key="score">0.998075</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23321" length="11"/>
<text>mutagenesis</text>
</annotation>
<annotation id="464">
<infon key="score">0.9994849</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23336" length="4"/>
<text>M196</text>
</annotation>
<annotation id="465">
<infon key="score">0.99947757</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23345" length="4"/>
<text>G199</text>
</annotation>
<annotation id="466">
<infon key="score">0.99952686</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:29Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23428" length="4"/>
<text>G197</text>
</annotation>
<annotation id="467">
<infon key="score">0.99806124</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23438" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="468">
<infon key="score">0.9982574</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23471" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="469">
<infon key="score">0.9896696</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:04:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23487" length="9"/>
<text>replacing</text>
</annotation>
<annotation id="470">
<infon key="score">0.99589795</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23500" length="7"/>
<text>alanine</text>
</annotation>
<annotation id="471">
<infon key="score">0.9990523</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23561" length="23"/>
<text>cofactor-binding pocket</text>
</annotation>
<annotation id="472">
<infon key="score">0.9981822</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23586" length="11"/>
<text>Mutagenesis</text>
</annotation>
<annotation id="473">
<infon key="score">0.9973271</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23606" length="7"/>
<text>glycine</text>
</annotation>
<annotation id="474">
<infon key="score">0.9975895</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:57:53Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23625" length="7"/>
<text>M.EcoKI</text>
</annotation>
<annotation id="475">
<infon key="score">0.991685</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:06Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23636" length="9"/>
<text>M.EcoP15I</text>
</annotation>
<annotation id="476">
<infon key="score">0.52265966</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23665" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="477">
<infon key="score">0.99767673</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:04:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23699" length="16"/>
<text>mutational study</text>
</annotation>
<annotation id="478">
<infon key="score">0.999522</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:33Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23743" length="4"/>
<text>F195</text>
</annotation>
<annotation id="479">
<infon key="score">0.99891686</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:08:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23797" length="5"/>
<text>F195A</text>
</annotation>
<annotation id="480">
<infon key="score">0.9988581</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23803" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="481">
<infon key="score">0.99871886</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:05:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23811" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="482">
<infon key="score">0.995095</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23875" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="483">
<infon key="score">0.9995129</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23910" length="4"/>
<text>F195</text>
</annotation>
<annotation id="484">
<infon key="score">0.99672353</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23937" length="22"/>
<text>π-stacking interaction</text>
</annotation>
<annotation id="485">
<infon key="score">0.99898654</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23984" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="486">
<infon key="score">0.9984652</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="24028" length="6"/>
<text>AdoMet</text>
</annotation>
<annotation id="487">
<infon key="score">0.8519888</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24035" length="8"/>
<text>bound in</text>
</annotation>
<annotation id="488">
<infon key="score">0.99786323</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:41Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24048" length="6"/>
<text>pocket</text>
</annotation>
<annotation id="655">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="24058" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="489">
<infon key="score">0.9978897</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24106" length="11"/>
<text>mutagenesis</text>
</annotation>
<annotation id="490">
<infon key="score">0.9979746</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:58:16Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24137" length="7"/>
<text>M.EcoRV</text>
</annotation>
<annotation id="491">
<infon key="score">0.9917243</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="24190" length="6"/>
<text>AdoMet</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>24211</offset>
<text>Potential DNA-binding sites</text>
<annotation id="492">
<infon key="score">0.99896115</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24221" length="17"/>
<text>DNA-binding sites</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>24239</offset>
<text>The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.</text>
<annotation id="493">
<infon key="score">0.9988854</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:39Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24252" length="18"/>
<text>DNA binding region</text>
</annotation>
<annotation id="494">
<infon key="score">0.8993177</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24274" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="807">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:29:18Z</infon>
<location offset="24297" length="12"/>
<text>hairpin loop</text>
</annotation>
<annotation id="495">
<infon key="score">0.997762</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24319" length="7"/>
<text>101-133</text>
</annotation>
<annotation id="744">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="24333" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="496">
<infon key="score">0.997704</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24347" length="7"/>
<text>136-166</text>
</annotation>
<annotation id="497">
<infon key="score">0.99897075</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24363" length="15"/>
<text>highly flexible</text>
</annotation>
<annotation id="792">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="24379" length="4"/>
<text>loop</text>
</annotation>
<annotation id="498">
<infon key="score">0.9976094</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:36Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24394" length="5"/>
<text>33-58</text>
</annotation>
<annotation id="808">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:29:18Z</infon>
<location offset="24406" length="12"/>
<text>hairpin loop</text>
</annotation>
<annotation id="805">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:57Z</infon>
<location offset="24427" length="2"/>
<text>β6</text>
</annotation>
<annotation id="803">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:43Z</infon>
<location offset="24434" length="2"/>
<text>β7</text>
</annotation>
<annotation id="499">
<infon key="score">0.9982666</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24460" length="9"/>
<text>conserved</text>
</annotation>
<annotation id="864">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:59:48Z</infon>
<location offset="24470" length="4"/>
<text>HRRY</text>
</annotation>
<annotation id="500">
<infon key="score">0.8395535</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24539" length="12"/>
<text>minor groove</text>
</annotation>
<annotation id="501">
<infon key="score">0.9990257</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:33:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24559" length="5"/>
<text>bound</text>
</annotation>
<annotation id="502">
<infon key="score">0.9979765</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="24565" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="745">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="24593" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="503">
<infon key="score">0.94398093</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24600" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="504">
<infon key="score">0.99851084</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24651" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="505">
<infon key="score">0.99915683</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24762" length="10"/>
<text>disordered</text>
</annotation>
<annotation id="746">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="24773" length="3"/>
<text>TRD</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>24784</offset>
<text>In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.</text>
<annotation id="506">
<infon key="score">0.99875844</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24801" length="15"/>
<text>highly flexible</text>
</annotation>
<annotation id="793">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="24817" length="4"/>
<text>loop</text>
</annotation>
<annotation id="782">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:27:09Z</infon>
<location offset="24848" length="4"/>
<text>DPPY</text>
</annotation>
<annotation id="507">
<infon key="score">0.66211873</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24862" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="508">
<infon key="score">0.9986974</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24894" length="16"/>
<text>electron density</text>
</annotation>
<annotation id="813">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:30:32Z</infon>
<location offset="24943" length="5"/>
<text>loops</text>
</annotation>
<annotation id="509">
<infon key="score">0.99905926</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24956" length="12"/>
<text>AdoMet-bound</text>
</annotation>
<annotation id="510">
<infon key="score">0.9975859</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24969" length="10"/>
<text>structures</text>
</annotation>
<annotation id="511">
<infon key="score">0.9956278</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:47:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24983" length="7"/>
<text>M.PvuII</text>
</annotation>
<annotation id="512">
<infon key="score">0.9992005</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:21:39Z</infon>
<infon key="identifier">PR:</infon>
<location offset="24992" length="4"/>
<text>DpnM</text>
</annotation>
<annotation id="513">
<infon key="score">0.99534625</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25000" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="794">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="25040" length="4"/>
<text>loop</text>
</annotation>
<annotation id="514">
<infon key="score">0.9867502</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:37:56Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25083" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="882">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:33:56Z</infon>
<location offset="25115" length="30"/>
<text>protein-DNA complex structures</text>
</annotation>
<annotation id="515">
<infon key="score">0.99605507</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25149" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="516">
<infon key="score">0.99500823</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:42Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25171" length="6"/>
<text>M.HhaI</text>
</annotation>
<annotation id="517">
<infon key="score">0.996617</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:50Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25196" length="8"/>
<text>M.HaeIII</text>
</annotation>
<annotation id="866">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:07Z</infon>
<location offset="25224" length="12"/>
<text>well-ordered</text>
</annotation>
<annotation id="795">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="25237" length="4"/>
<text>loop</text>
</annotation>
<annotation id="518">
<infon key="score">0.99599034</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="25251" length="10"/>
<text>structures</text>
</annotation>
<annotation id="519">
<infon key="score">0.7158421</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25293" length="7"/>
<text>adenine</text>
</annotation>
<annotation id="520">
<infon key="score">0.9972713</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:48:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25311" length="13"/>
<text>hydrogen bond</text>
</annotation>
<annotation id="796">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="25399" length="4"/>
<text>loop</text>
</annotation>
<annotation id="521">
<infon key="score">0.9991862</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25413" length="6"/>
<text>MTases</text>
</annotation>
<annotation id="656">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="25426" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>25548</offset>
<text>Key residue for wider spectrum of substrate recognition</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>25604</offset>
<text>Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.</text>
<annotation id="522">
<infon key="score">0.6295532</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25637" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="523">
<infon key="score">0.9984703</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="25679" length="16"/>
<text>N6 adenine MTase</text>
</annotation>
<annotation id="524">
<infon key="score">0.989359</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25719" length="7"/>
<text>adenine</text>
</annotation>
<annotation id="525">
<infon key="score">0.9972758</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:21Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25730" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="526">
<infon key="score">0.9922677</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:24Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25741" length="10"/>
<text>5′-GGAG-3′</text>
</annotation>
<annotation id="527">
<infon key="score">0.9940481</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:07:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25764" length="8"/>
<text>adenines</text>
</annotation>
<annotation id="528">
<infon key="score">0.99715126</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:05Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25776" length="10"/>
<text>5′-GAAG-3′</text>
</annotation>
<annotation id="529">
<infon key="score">0.99270916</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="25862" length="7"/>
<text>adenine</text>
</annotation>
<annotation id="530">
<infon key="score">0.9974624</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:26Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="25873" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="657">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="25899" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="867">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:01:59Z</infon>
<location offset="25943" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="658">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="25988" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="531">
<infon key="score">0.99622726</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26025" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="532">
<infon key="score">0.9988635</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26047" length="27"/>
<text>multiple sequence alignment</text>
</annotation>
<annotation id="533">
<infon key="score">0.9983783</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:05:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26085" length="19"/>
<text>sequence comparison</text>
</annotation>
<annotation id="534">
<infon key="score">0.9984338</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:05:10Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26109" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="535">
<infon key="score">0.9994925</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26154" length="3"/>
<text>P41</text>
</annotation>
<annotation id="536">
<infon key="score">0.99946815</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26159" length="4"/>
<text>N111</text>
</annotation>
<annotation id="537">
<infon key="score">0.9995011</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26165" length="4"/>
<text>K165</text>
</annotation>
<annotation id="538">
<infon key="score">0.99950707</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26174" length="4"/>
<text>T166</text>
</annotation>
<annotation id="539">
<infon key="score">0.9943739</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:05:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26197" length="8"/>
<text>replaced</text>
</annotation>
<annotation id="540">
<infon key="score">0.99705803</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26209" length="6"/>
<text>serine</text>
</annotation>
<annotation id="541">
<infon key="score">0.997026</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26217" length="9"/>
<text>threonine</text>
</annotation>
<annotation id="542">
<infon key="score">0.9970084</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26228" length="9"/>
<text>threonine</text>
</annotation>
<annotation id="543">
<infon key="score">0.9967733</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:02:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26242" length="6"/>
<text>valine</text>
</annotation>
<annotation id="869">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:01Z</infon>
<location offset="26284" length="29"/>
<text>[3H]AdoMet radiological assay</text>
</annotation>
<annotation id="857">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="26342" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="544">
<infon key="score">0.99777615</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:34:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26374" length="9"/>
<text>wide type</text>
</annotation>
<annotation id="545">
<infon key="score">0.98206836</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26400" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="868">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:08Z</infon>
<location offset="26450" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="546">
<infon key="score">0.99718624</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:15Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26463" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="547">
<infon key="score">0.9764722</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26477" length="11"/>
<text>5′-GAAG-3′,</text>
</annotation>
<annotation id="548">
<infon key="score">0.9746132</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26497" length="7"/>
<text>mutants</text>
</annotation>
<annotation id="858">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="26538" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="549">
<infon key="score">0.9992687</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26579" length="2"/>
<text>wt</text>
</annotation>
<annotation id="659">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="26582" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="550">
<infon key="score">0.9540184</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:02:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26631" length="11"/>
<text>5′-GGAG-3′,</text>
</annotation>
<annotation id="859">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="26647" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="551">
<infon key="score">0.99837804</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:42Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26679" length="4"/>
<text>P41S</text>
</annotation>
<annotation id="552">
<infon key="score">0.99891484</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26684" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="553">
<infon key="score">0.9990494</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:55:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="26733" length="9"/>
<text>wild type</text>
</annotation>
<annotation id="660">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="26743" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
</passage>
<passage>
<infon key="file">oncotarget-07-40965-g005.jpg</infon>
<infon key="id">F5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>26782</offset>
<text>Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI</text>
<annotation id="554">
<infon key="score">0.99885774</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:45:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26782" length="18"/>
<text>Sequence alignment</text>
</annotation>
<annotation id="555">
<infon key="score">0.99821293</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26802" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="556">
<infon key="score">0.97248703</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:22Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26826" length="36"/>
<text>radioactive methyl transfer activity</text>
</annotation>
<annotation id="661">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="26921" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
</passage>
<passage>
<infon key="file">oncotarget-07-40965-g005.jpg</infon>
<infon key="id">F5</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>26931</offset>
<text>A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background.</text>
<annotation id="557">
<infon key="score">0.9984727</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:45:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26934" length="18"/>
<text>Sequence alignment</text>
</annotation>
<annotation id="662">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="26956" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="558">
<infon key="score">0.997293</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="26974" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="559">
<infon key="score">0.99947685</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27052" length="3"/>
<text>P41</text>
</annotation>
<annotation id="560">
<infon key="score">0.99948114</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27057" length="4"/>
<text>N111</text>
</annotation>
<annotation id="561">
<infon key="score">0.99947375</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:03:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27063" length="4"/>
<text>K165</text>
</annotation>
<annotation id="562">
<infon key="score">0.9994906</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27072" length="4"/>
<text>T166</text>
</annotation>
<annotation id="663">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="27080" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="563">
<infon key="score">0.39663774</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27102" length="5"/>
<text>26695</text>
</annotation>
<annotation id="564">
<infon key="score">0.99588466</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:04:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27129" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="565">
<infon key="score">0.9985169</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:45:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27153" length="18"/>
<text>sequence alignment</text>
</annotation>
<annotation id="566">
<infon key="score">0.8075491</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:07:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27236" length="7"/>
<text>WebLogo</text>
</annotation>
<annotation id="860">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="27277" length="6"/>
<text>Methyl</text>
</annotation>
<annotation id="567">
<infon key="score">0.998982</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:49:41Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27324" length="2"/>
<text>wt</text>
</annotation>
<annotation id="664">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="27327" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="870">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:05:35Z</infon>
<location offset="27338" length="14"/>
<text>M1.HpyAVI-P41S</text>
</annotation>
<annotation id="871">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:05:53Z</infon>
<location offset="27354" length="15"/>
<text>M1.HpyAVI-N111T</text>
</annotation>
<annotation id="873">
<infon key="type">mutant</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:06:47Z</infon>
<location offset="27375" length="21"/>
<text>M1.HpyAVI-K165R T166V</text>
</annotation>
<annotation id="568">
<infon key="score">0.9894895</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:38Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27455" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="872">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:06:14Z</infon>
<location offset="27470" length="10"/>
<text>5′-GAGG-3′</text>
</annotation>
<annotation id="569">
<infon key="score">0.7406915</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:06:17Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27482" length="10"/>
<text>5′-GAAG-3′</text>
</annotation>
<annotation id="570">
<infon key="score">0.8041685</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:06:19Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27496" length="10"/>
<text>5′-GGAG-3′</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>27668</offset>
<text>Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.</text>
<annotation id="571">
<infon key="score">0.9995059</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27701" length="3"/>
<text>P41</text>
</annotation>
<annotation id="572">
<infon key="score">0.9565232</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:07:25Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27753" length="4"/>
<text>GGAG</text>
</annotation>
<annotation id="665">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="27761" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="573">
<infon key="score">0.998413</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27803" length="15"/>
<text>highly flexible</text>
</annotation>
<annotation id="797">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="27819" length="4"/>
<text>loop</text>
</annotation>
<annotation id="574">
<infon key="score">0.95510674</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:42Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27841" length="9"/>
<text>33 and 58</text>
</annotation>
<annotation id="874">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:07:49Z</infon>
<location offset="27873" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="575">
<infon key="score">0.99823356</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:14Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27927" length="11"/>
<text>Replacement</text>
</annotation>
<annotation id="576">
<infon key="score">0.99676514</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27942" length="6"/>
<text>serine</text>
</annotation>
<annotation id="798">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:01Z</infon>
<location offset="28081" length="4"/>
<text>loop</text>
</annotation>
<annotation id="577">
<infon key="score">0.5137013</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28186" length="5"/>
<text>26695</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">title_1</infon>
<offset>28193</offset>
<text>DISCUSSION</text>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>28204</offset>
<text>Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.</text>
<annotation id="578">
<infon key="score">0.99879885</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28217" length="9"/>
<text>DNA-bound</text>
</annotation>
<annotation id="579">
<infon key="score">0.9980544</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:30:25Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28227" length="9"/>
<text>structure</text>
</annotation>
<annotation id="580">
<infon key="score">0.9987933</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28268" length="24"/>
<text>γ-class N6-adenine MTase</text>
</annotation>
<annotation id="581">
<infon key="score">0.69096255</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28318" length="7"/>
<text>adenine</text>
</annotation>
<annotation id="582">
<infon key="score">0.9610856</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28345" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="861">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="28371" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="583">
<infon key="score">0.9975112</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:55:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28471" length="16"/>
<text>N6-methyladenine</text>
</annotation>
<annotation id="584">
<infon key="score">0.99863404</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28496" length="10"/>
<text>eukaryotic</text>
</annotation>
<annotation id="585">
<infon key="score">0.9988544</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:05:55Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28553" length="17"/>
<text>N6-adenine MTases</text>
</annotation>
<annotation id="586">
<infon key="score">0.99871874</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28591" length="10"/>
<text>eukaryotes</text>
</annotation>
<annotation id="587">
<infon key="score">0.99865556</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:08:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28603" length="43"/>
<text>Biochemical and structural characterization</text>
</annotation>
<annotation id="588">
<infon key="score">0.9822526</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28650" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="862">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:56:49Z</infon>
<location offset="28700" length="6"/>
<text>methyl</text>
</annotation>
<annotation id="589">
<infon key="score">0.99716395</infon>
<infon key="type">ptm</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:55:25Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28752" length="16"/>
<text>N6-methyladenine</text>
</annotation>
<annotation id="590">
<infon key="score">0.9985537</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28772" length="10"/>
<text>eukaryotes</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>28788</offset>
<text>Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.</text>
<annotation id="591">
<infon key="score">0.998248</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28808" length="10"/>
<text>DNA MTases</text>
</annotation>
<annotation id="592">
<infon key="score">0.9987644</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28840" length="7"/>
<text>monomer</text>
</annotation>
<annotation id="666">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="28883" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="593">
<infon key="score">0.998727</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28905" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="594">
<infon key="score">0.9870143</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="28919" length="7"/>
<text>crystal</text>
</annotation>
<annotation id="595">
<infon key="score">0.99645835</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28967" length="28"/>
<text>β-class DNA exocyclic MTases</text>
</annotation>
<annotation id="596">
<infon key="score">0.9813897</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29031" length="7"/>
<text>crystal</text>
</annotation>
<annotation id="597">
<infon key="score">0.9987569</infon>
<infon key="type">oligomeric_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:49:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29072" length="5"/>
<text>dimer</text>
</annotation>
<annotation id="598">
<infon key="score">0.9984802</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:51:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29130" length="10"/>
<text>DNA MTases</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>29145</offset>
<text>The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.</text>
<annotation id="599">
<infon key="score">0.9987935</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29149" length="15"/>
<text>highly flexible</text>
</annotation>
<annotation id="600">
<infon key="score">0.99759173</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29182" length="5"/>
<text>33-58</text>
</annotation>
<annotation id="747">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="29193" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="601">
<infon key="score">0.9977372</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:37Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29207" length="7"/>
<text>133-163</text>
</annotation>
<annotation id="602">
<infon key="score">0.76505375</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29219" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="603">
<infon key="score">0.9985341</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:38:58Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29259" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="875">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:09:53Z</infon>
<location offset="29266" length="23"/>
<text>minor and major grooves</text>
</annotation>
<annotation id="604">
<infon key="score">0.9994406</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:16Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29412" length="3"/>
<text>P41</text>
</annotation>
<annotation id="667">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="29487" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>29498</offset>
<text>The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.</text>
<annotation id="605">
<infon key="score">0.66722006</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:34:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29502" length="7"/>
<text>missing</text>
</annotation>
<annotation id="799">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:28:02Z</infon>
<location offset="29510" length="4"/>
<text>loop</text>
</annotation>
<annotation id="606">
<infon key="score">0.71347064</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:06:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29532" length="9"/>
<text>33 and 58</text>
</annotation>
<annotation id="607">
<infon key="score">0.9982004</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:39:02Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29551" length="3"/>
<text>DNA</text>
</annotation>
<annotation id="608">
<infon key="score">0.9979321</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:34:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29579" length="6"/>
<text>stable</text>
</annotation>
<annotation id="609">
<infon key="score">0.99098516</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:01:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29637" length="6"/>
<text>M.TaqI</text>
</annotation>
<annotation id="610">
<infon key="score">0.9987587</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:10:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29645" length="15"/>
<text>Crystallization</text>
</annotation>
<annotation id="611">
<infon key="score">0.96516407</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:10:16Z</infon>
<infon key="identifier">GO:</infon>
<location offset="29664" length="13"/>
<text>M1.HpyAVI-DNA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>29817</offset>
<text>DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.</text>
<annotation id="673">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:29Z</infon>
<location offset="29817" length="15"/>
<text>DNA methylation</text>
</annotation>
<annotation id="612">
<infon key="score">0.9985331</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29860" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="877">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:11:03Z</infon>
<location offset="29885" length="23"/>
<text>DNA adenine methylation</text>
</annotation>
<annotation id="613">
<infon key="score">0.9984968</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:48:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29973" length="8"/>
<text>bacteria</text>
</annotation>
<annotation id="614">
<infon key="score">0.9971612</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29992" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="876">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:11:02Z</infon>
<location offset="30016" length="23"/>
<text>DNA adenine methylation</text>
</annotation>
<annotation id="615">
<infon key="score">0.99845654</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:37:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30091" length="29"/>
<text>DNA adenine methyltransferase</text>
</annotation>
<annotation id="674">
<infon key="type">ptm</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:29Z</infon>
<location offset="30163" length="15"/>
<text>DNA methylation</text>
</annotation>
<annotation id="616">
<infon key="score">0.99820757</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:53:03Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30199" length="9"/>
<text>bacterial</text>
</annotation>
<annotation id="617">
<infon key="score">0.7532001</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T10:05:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30219" length="11"/>
<text>Knockout of</text>
</annotation>
<annotation id="668">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="30231" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="618">
<infon key="score">0.9971716</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30272" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="619">
<infon key="score">0.99724466</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30296" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="620">
<infon key="score">0.9974534</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30437" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="621">
<infon key="score">0.9974273</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30481" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="622">
<infon key="score">0.99812746</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:58:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30642" length="14"/>
<text>adenine MTases</text>
</annotation>
<annotation id="669">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<location offset="30665" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="623">
<infon key="score">0.9971631</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T15:04:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30718" length="9"/>
<text>H. pylori</text>
</annotation>
<annotation id="624">
<infon key="score">0.9993161</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30800" length="3"/>
<text>D29</text>
</annotation>
<annotation id="625">
<infon key="score">0.99932635</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30807" length="4"/>
<text>E216</text>
</annotation>
<annotation id="626">
<infon key="score">0.9974186</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30830" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="627">
<infon key="score">0.9989734</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:11:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30881" length="16"/>
<text>highly conserved</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>30950</offset>
<text>In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.</text>
<annotation id="628">
<infon key="score">0.9974125</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:12:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30966" length="9"/>
<text>structure</text>
</annotation>
<annotation id="629">
<infon key="score">0.8668125</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:49:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30979" length="9"/>
<text>M1.HpyAVI</text>
</annotation>
<annotation id="630">
<infon key="score">0.9987343</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:00:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31008" length="10"/>
<text>disordered</text>
</annotation>
<annotation id="748">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T08:23:59Z</infon>
<location offset="31019" length="3"/>
<text>TRD</text>
</annotation>
<annotation id="884">
<infon key="type">residue_name_number</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:36:37Z</infon>
<location offset="31041" length="3"/>
<text>P41</text>
</annotation>
<annotation id="631">
<infon key="score">0.9987941</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:35:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31073" length="18"/>
<text>DNA binding region</text>
</annotation>
<annotation id="632">
<infon key="score">0.9995447</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31158" length="3"/>
<text>D29</text>
</annotation>
<annotation id="633">
<infon key="score">0.9995259</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:50:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31166" length="4"/>
<text>E216</text>
</annotation>
<annotation id="634">
<infon key="score">0.9988655</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:12:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="31244" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="635">
<infon key="score">0.9980763</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:27:09Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31265" length="16"/>
<text>N6-adenine MTase</text>
</annotation>
<annotation id="636">
<infon key="score">0.9978216</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31285" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="637">
<infon key="score">0.99823475</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-18T09:25:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31394" length="8"/>
<text>H.pylori</text>
</annotation>
<annotation id="638">
<infon key="score">0.99853706</infon>
<infon key="type">species</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:54:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="31406" length="5"/>
<text>human</text>
</annotation>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>31413</offset>
<text>MATERIALS AND METHODS</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>31435</offset>
<text>Protein expression and purification</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>31471</offset>
<text>The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32157</offset>
<text>Crystallization and data collection</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32193</offset>
<text>Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>32841</offset>
<text>Structure determination and refinement</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>32880</offset>
<text>The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>33569</offset>
<text>Detection of protein dimerization</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>33603</offset>
<text>The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>33779</offset>
<text>The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>34019</offset>
<text>Binding affinity quantification via microscale thermophoresis (MST)</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34087</offset>
<text>Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>34892</offset>
<text>Radioactive methyltransferase analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34931</offset>
<text>Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate.</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>35753</offset>
<text>SUPPLEMENTARY FIGURES AND TABLES</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>35786</offset>
<text>CONFLICTS OF INTEREST</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>35808</offset>
<text>The authors declare that they have no conflicts of interest.</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>35869</offset>
<text>ACCESSION CODES</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>35885</offset>
<text>Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ respectively.</text>
</passage>
<passage>
<infon key="section_type">REF</infon>
<infon key="type">title</infon>
<offset>36054</offset>
<text>REFERENCES</text>
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<offset>36065</offset>
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<infon key="section_type">TITLE</infon>
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<text>Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40</text>
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<text>Roquin</text>
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<text>hexaloop</text>
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<text>3′-UTR</text>
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<text>Ox40</text>
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<infon key="section_type">ABSTRACT</infon>
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<text>The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.</text>
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<text>RNA-binding protein</text>
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<text>Roquin</text>
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<text>Roquin</text>
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<text>costimulatory receptors</text>
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<text>tumor necrosis factor receptor superfamily 4</text>
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<text>Tnfrs4</text>
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<text>Ox40</text>
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<text>CDE</text>
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<text>triloop hairpin</text>
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<text>U-rich hexaloop motif</text>
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<text>alternative decay element</text>
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<infon key="updated_at">2023-09-15T12:09:19Z</infon>
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<location offset="572" length="3"/>
<text>ADE</text>
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<text>Roquin-1</text>
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<location offset="633" length="3"/>
<text>ROQ</text>
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<text>ADE</text>
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<location offset="700" length="3"/>
<text>ADE</text>
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<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="844" length="4"/>
<text>Ox40</text>
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<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<text>hexaloop cis elements</text>
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<infon key="identifier">CHEBI:</infon>
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<text>messenger RNAs</text>
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<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<infon key="section_type">ABSTRACT</infon>
<infon key="type">abstract</infon>
<offset>1004</offset>
<text> Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.</text>
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<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<infon key="updated_at">2023-09-15T12:18:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="1018" length="19"/>
<text>RNA-binding protein</text>
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<annotation id="35">
<infon key="score">0.9930662</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1109" length="4"/>
<text>Ox40</text>
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<annotation id="36">
<infon key="score">0.99891186</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>RNA</text>
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<annotation id="37">
<infon key="score">0.99862254</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:26Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1149" length="9"/>
<text>structure</text>
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<annotation id="1098">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:14Z</infon>
<location offset="1184" length="25"/>
<text>alternative decay element</text>
</annotation>
<annotation id="38">
<infon key="score">0.9992798</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1254" length="6"/>
<text>Roquin</text>
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<annotation id="39">
<infon key="score">0.9988141</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:28:13Z</infon>
<infon key="identifier">MESH:</infon>
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<text>structural and biochemical techniques</text>
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</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>1306</offset>
<text>Posttranscriptional gene regulation is involved in a wide range of cellular functions and its critical importance has been described for many developmental and differentiation processes. Consistently, mutations of factors involved in posttranscriptional gene regulation pathways were found associated with a number of genetically inherited diseases. The Roquin protein is essential in T cells for the prevention of autoimmune disease. This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1660" length="6"/>
<text>Roquin</text>
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<annotation id="41">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1797" length="8"/>
<text>Roquin-1</text>
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<annotation id="42">
<infon key="score">0.9994506</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1841" length="6"/>
<text>Met199</text>
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<annotation id="43">
<infon key="score">0.9090607</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="1851" length="3"/>
<text>Arg</text>
</annotation>
<annotation id="44">
<infon key="score">0.9979857</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="1943" length="4"/>
<text>mice</text>
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<annotation id="45">
<infon key="score">0.9948087</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:18Z</infon>
<infon key="identifier">GENE:</infon>
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<text>Rc3h1</text>
</annotation>
<annotation id="46">
<infon key="score">0.9945793</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:23Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="1963" length="5"/>
<text>Rc3h2</text>
</annotation>
<annotation id="47">
<infon key="score">0.99835825</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="1989" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="48">
<infon key="score">0.99819595</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2002" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="49">
<infon key="score">0.99857664</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2023" length="11"/>
<text>vertebrates</text>
</annotation>
<annotation id="50">
<infon key="score">0.99825436</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2107" length="4"/>
<text>mice</text>
</annotation>
<annotation id="51">
<infon key="score">0.9137891</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:53Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2220" length="11"/>
<text>deletion of</text>
</annotation>
<annotation id="52">
<infon key="score">0.99057406</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2232" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="53">
<infon key="score">0.9987157</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2370" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="54">
<infon key="score">0.9979331</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:51Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2451" length="24"/>
<text>co-stimulatory receptors</text>
</annotation>
<annotation id="55">
<infon key="score">0.99867827</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2484" length="4"/>
<text>Icos</text>
</annotation>
<annotation id="56">
<infon key="score">0.99837804</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2490" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="57">
<infon key="score">0.9986387</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:53:21Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2499" length="6"/>
<text>CTLA-4</text>
</annotation>
<annotation id="58">
<infon key="score">0.9791268</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:13:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2511" length="9"/>
<text>cytokines</text>
</annotation>
<annotation id="59">
<infon key="score">0.91296864</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:14:28Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2529" length="18"/>
<text>interleukin (IL)-6</text>
</annotation>
<annotation id="1157">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:15:54Z</infon>
<location offset="2552" length="22"/>
<text>tumour necrosis factor</text>
</annotation>
<annotation id="60">
<infon key="score">0.9902424</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="2582" length="21"/>
<text>transcription factors</text>
</annotation>
<annotation id="61">
<infon key="score">0.9894326</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:06Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2612" length="4"/>
<text>IRF4</text>
</annotation>
<annotation id="62">
<infon key="score">0.65273774</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2618" length="5"/>
<text>IκBNS</text>
</annotation>
<annotation id="63">
<infon key="score">0.963164</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:15Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2628" length="4"/>
<text>IκBζ</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>2652</offset>
<text>We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.</text>
<annotation id="64">
<infon key="score">0.9882385</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:32Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2678" length="30"/>
<text>structural and functional data</text>
</annotation>
<annotation id="65">
<infon key="score">0.99861914</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2716" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="66">
<infon key="score">0.9994229</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2725" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="67">
<infon key="score">0.9990214</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="2736" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="68">
<infon key="score">0.9817135</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2757" length="26"/>
<text>constitutive decay element</text>
</annotation>
<annotation id="69">
<infon key="score">0.94088954</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2785" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="70">
<infon key="score">0.9986866</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2793" length="15"/>
<text>short stem loop</text>
</annotation>
<annotation id="71">
<infon key="score">0.99715364</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2810" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1237">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<location offset="2844" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="72">
<infon key="score">0.99731666</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2863" length="23"/>
<text>3′-untranslated regions</text>
</annotation>
<annotation id="73">
<infon key="score">0.99472845</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2888" length="7"/>
<text>3′-UTRs</text>
</annotation>
<annotation id="74">
<infon key="score">0.9919091</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:01Z</infon>
<infon key="identifier">PR:</infon>
<location offset="2921" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="75">
<infon key="score">0.9994338</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2936" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="76">
<infon key="score">0.9923204</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="2957" length="26"/>
<text>extended winged helix fold</text>
</annotation>
<annotation id="77">
<infon key="score">0.9763267</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3041" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="78">
<infon key="score">0.9922246</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3103" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="79">
<infon key="score">0.8482103</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3107" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="80">
<infon key="score">0.9985152</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3111" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="81">
<infon key="score">0.99448186</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3120" length="15"/>
<text>structural data</text>
</annotation>
<annotation id="82">
<infon key="score">0.983335</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3140" length="19"/>
<text>mutational analysis</text>
</annotation>
<annotation id="83">
<infon key="score">0.9987087</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3236" length="4"/>
<text>loop</text>
</annotation>
<annotation id="84">
<infon key="score">0.9761445</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3245" length="4"/>
<text>stem</text>
</annotation>
<annotation id="85">
<infon key="score">0.91149104</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3257" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="86">
<infon key="score">0.9992151</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3300" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="87">
<infon key="score">0.9987693</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3351" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="1065">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="3432" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="1160">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="3441" length="2"/>
<text>SL</text>
</annotation>
<annotation id="88">
<infon key="score">0.9989596</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3460" length="19"/>
<text>second binding site</text>
</annotation>
<annotation id="89">
<infon key="score">0.9989705</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:43Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3486" length="19"/>
<text>double-stranded RNA</text>
</annotation>
<annotation id="90">
<infon key="score">0.99912053</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:48Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="3507" length="5"/>
<text>dsRNA</text>
</annotation>
<annotation id="91">
<infon key="score">0.9981774</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="3524" length="8"/>
<text>extended</text>
</annotation>
<annotation id="92">
<infon key="score">0.9983943</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3533" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="1066">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="3570" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="93">
<infon key="score">0.9977644</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:10Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3593" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="94">
<infon key="score">0.9992968</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="3602" length="3"/>
<text>ROQ</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>3652</offset>
<text>We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.</text>
<annotation id="95">
<infon key="score">0.99875194</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3702" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="96">
<infon key="score">0.99869066</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3715" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="97">
<infon key="score">0.9988292</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="3761" length="11"/>
<text>paracaspase</text>
</annotation>
<annotation id="98">
<infon key="score">0.9991509</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:18Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3773" length="5"/>
<text>MALT1</text>
</annotation>
<annotation id="99">
<infon key="score">0.998901</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:18Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3796" length="5"/>
<text>MALT1</text>
</annotation>
<annotation id="100">
<infon key="score">0.999226</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="3841" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="101">
<infon key="score">0.99925715</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4182" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="102">
<infon key="score">0.9953003</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4241" length="16"/>
<text>binding affinity</text>
</annotation>
<annotation id="1161">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="4276" length="2"/>
<text>SL</text>
</annotation>
<annotation id="103">
<infon key="score">0.99862415</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4295" length="7"/>
<text>3′-UTRs</text>
</annotation>
<annotation id="104">
<infon key="score">0.9908908</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4313" length="5"/>
<text>mRNAs</text>
</annotation>
<annotation id="105">
<infon key="score">0.9993056</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4390" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="1162">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="4411" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1238">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<location offset="4414" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="106">
<infon key="score">0.9992386</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4452" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="107">
<infon key="score">0.99929154</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4546" length="6"/>
<text>Roquin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">INTRO</infon>
<infon key="type">paragraph</infon>
<offset>4622</offset>
<text>Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.</text>
<annotation id="108">
<infon key="score">0.951145</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="4710" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="109">
<infon key="score">0.9992292</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4745" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="1264">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:33Z</infon>
<location offset="4781" length="15"/>
<text>U-rich hexaloop</text>
</annotation>
<annotation id="1163">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="4797" length="2"/>
<text>SL</text>
</annotation>
<annotation id="110">
<infon key="score">0.99834704</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4807" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="111">
<infon key="score">0.99911016</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4817" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="112">
<infon key="score">0.99903107</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="4822" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="113">
<infon key="score">0.9978402</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4835" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="114">
<infon key="score">0.9993236</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:52Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4844" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="115">
<infon key="score">0.9987203</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="4890" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1067">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="4929" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="116">
<infon key="score">0.9922536</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:24:00Z</infon>
<infon key="identifier">SO:</infon>
<location offset="4942" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="1164">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="4951" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1274">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:23Z</infon>
<location offset="4954" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="117">
<infon key="score">0.9987448</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="4984" length="57"/>
<text>Systematic Evolution of Ligands by Exponential Enrichment</text>
</annotation>
<annotation id="118">
<infon key="score">0.9978467</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:10:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5043" length="5"/>
<text>SELEX</text>
</annotation>
<annotation id="119">
<infon key="score">0.91901964</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:43Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5067" length="22"/>
<text>X-ray crystallographic</text>
</annotation>
<annotation id="120">
<infon key="score">0.9959416</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5091" length="3"/>
<text>NMR</text>
</annotation>
<annotation id="121">
<infon key="score">0.8331451</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5096" length="31"/>
<text>biochemical and functional data</text>
</annotation>
<annotation id="122">
<infon key="score">0.98718065</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:48Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5142" length="19"/>
<text>mutational analysis</text>
</annotation>
<annotation id="123">
<infon key="score">0.8578007</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5184" length="7"/>
<text>triloop</text>
</annotation>
<annotation id="124">
<infon key="score">0.9144711</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="5196" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="1165">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="5205" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1273">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:22:53Z</infon>
<location offset="5208" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="125">
<infon key="score">0.9992194</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:55Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5254" length="6"/>
<text>Roquin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_1</infon>
<offset>5275</offset>
<text>Results</text>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>5283</offset>
<text>SELEX identifies novel RNA ligands of Roquin-1</text>
<annotation id="126">
<infon key="score">0.99852544</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:10:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="5283" length="5"/>
<text>SELEX</text>
</annotation>
<annotation id="127">
<infon key="score">0.996561</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5306" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="128">
<infon key="score">0.9990085</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="5321" length="8"/>
<text>Roquin-1</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>5330</offset>
<text>We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. Enriched sequences were clustered into so-called patterns with highly homologous sequences. Hereby, the algorithm at first identified frequent motifs of five to eight nucleotides length and subsequently used iterative cycles of proto-pattern formation to cluster sequences bearing two of such frequent motifs. A final aptamer pattern was built up by sequences bearing two frequent motifs and, at the same time, having high similarities also in other sequence parts. Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). Notably, our SELEX approach did not reveal the previously identified CDE sequence. We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).</text>
<annotation id="129">
<infon key="score">0.9651447</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:25:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="5353" length="12"/>
<text>Roquin-bound</text>
</annotation>
<annotation id="130">
<infon key="score">0.9924872</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:52Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="5366" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="131">
<infon key="score">0.99846774</infon>
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<text>We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).</text>
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<text>Ox40</text>
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<text>SL</text>
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<infon key="updated_at">2023-09-15T12:11:00Z</infon>
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<text>NMR</text>
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<text>stem regions</text>
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<location offset="9087" length="17"/>
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<text>SL</text>
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<text>RNAs</text>
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<location offset="9257" length="3"/>
<text>ADE</text>
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<text>CSPs</text>
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<text>RNA</text>
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<location offset="9724" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>bound to</text>
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<annotation id="1108">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="10049" length="2"/>
<text>SL</text>
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<annotation id="1276">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="10052" length="4"/>
<text>RNAs</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>10057</offset>
<text>To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10074" length="6"/>
<text>Roquin</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="10105" length="2"/>
<text>SL</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:10:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10135" length="5"/>
<text>SELEX</text>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="10154" length="6"/>
<text>solved</text>
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<annotation id="210">
<infon key="score">0.9981201</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:35Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10161" length="18"/>
<text>crystal structures</text>
</annotation>
<annotation id="211">
<infon key="score">0.99574417</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:35Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10187" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="212">
<infon key="score">0.99937755</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10196" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="213">
<infon key="score">0.9989785</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10207" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="214">
<infon key="score">0.9867727</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10236" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="215">
<infon key="score">0.99827456</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10254" length="10"/>
<text>structures</text>
</annotation>
<annotation id="216">
<infon key="score">0.99751127</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10268" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="217">
<infon key="score">0.9990022</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10272" length="8"/>
<text>bound to</text>
</annotation>
<annotation id="218">
<infon key="score">0.9742184</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10292" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="219">
<infon key="score">0.8739459</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10296" length="2"/>
<text>SL</text>
</annotation>
<annotation id="220">
<infon key="score">0.9306097</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10341" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1109">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="10346" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1183">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="10355" length="2"/>
<text>SL</text>
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<annotation id="1277">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="10358" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="221">
<infon key="score">0.99819595</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="10442" length="10"/>
<text>structures</text>
</annotation>
<annotation id="222">
<infon key="score">0.99550533</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10457" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="1184">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="10471" length="2"/>
<text>SL</text>
</annotation>
<annotation id="223">
<infon key="score">0.9942965</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10490" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="224">
<infon key="score">0.99924064</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10557" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="225">
<infon key="score">0.9994573</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10561" length="5"/>
<text>helix</text>
</annotation>
<annotation id="226">
<infon key="score">0.99950755</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10567" length="2"/>
<text>α4</text>
</annotation>
<annotation id="227">
<infon key="score">0.9995253</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10597" length="2"/>
<text>β3</text>
</annotation>
<annotation id="228">
<infon key="score">0.99905974</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10645" length="5"/>
<text>dsRNA</text>
</annotation>
<annotation id="229">
<infon key="score">0.8476505</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10651" length="4"/>
<text>stem</text>
</annotation>
<annotation id="230">
<infon key="score">0.54066336</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="10717" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="231">
<infon key="score">0.938291</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10721" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="232">
<infon key="score">0.6966292</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10725" length="2"/>
<text>SL</text>
</annotation>
<annotation id="233">
<infon key="score">0.96888745</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10728" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="234">
<infon key="score">0.99511886</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:49:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10802" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="235">
<infon key="score">0.99347335</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:37:18Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10847" length="7"/>
<text>triloop</text>
</annotation>
<annotation id="236">
<infon key="score">0.9789211</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10862" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="237">
<infon key="score">0.873177</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="10866" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="238">
<infon key="score">0.9607949</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10954" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="239">
<infon key="score">0.7767095</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10958" length="2"/>
<text>SL</text>
</annotation>
<annotation id="240">
<infon key="score">0.8281227</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="10965" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1185">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="10974" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1278">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="10977" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="241">
<infon key="score">0.9970162</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11009" length="10"/>
<text>structures</text>
</annotation>
<annotation id="1239">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="11032" length="3"/>
<text>RNA</text>
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<annotation id="242">
<infon key="score">0.99493295</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:03:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11125" length="3"/>
<text>C19</text>
</annotation>
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<infon key="score">0.5360428</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11129" length="5"/>
<text>bulge</text>
</annotation>
<annotation id="1304">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:44Z</infon>
<location offset="11140" length="16"/>
<text>non-Watson–Crick</text>
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<annotation id="244">
<infon key="score">0.99560696</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:03:54Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11157" length="2"/>
<text>G6</text>
</annotation>
<annotation id="245">
<infon key="score">0.96903014</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11160" length="3"/>
<text>G15</text>
</annotation>
<annotation id="1305">
<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:56Z</infon>
<location offset="11164" length="9"/>
<text>base pair</text>
</annotation>
<annotation id="246">
<infon key="score">0.9992611</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:03:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11197" length="2"/>
<text>U1</text>
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<infon key="score">0.9995658</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11205" length="6"/>
<text>Trp184</text>
</annotation>
<annotation id="248">
<infon key="score">0.99956375</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11216" length="6"/>
<text>Phe194</text>
</annotation>
<annotation id="249">
<infon key="score">0.7797527</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11230" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1186">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="11239" length="2"/>
<text>SL</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="11242" length="3"/>
<text>RNA</text>
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<infon key="score">0.99824846</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:35:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11357" length="9"/>
<text>structure</text>
</annotation>
<annotation id="251">
<infon key="score">0.8127755</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11374" length="4"/>
<text>Ox40</text>
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<annotation id="1110">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="11379" length="3"/>
<text>ADE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="11388" length="2"/>
<text>SL</text>
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<annotation id="1241">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="11391" length="3"/>
<text>RNA</text>
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<annotation id="252">
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<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11426" length="4"/>
<text>Ox40</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
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<location offset="11431" length="6"/>
<text>3′-UTR</text>
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<passage>
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<infon key="type">paragraph</infon>
<offset>11492</offset>
<text>The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.</text>
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<infon key="score">0.9986315</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:49:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11539" length="20"/>
<text>double-stranded stem</text>
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<annotation id="255">
<infon key="score">0.68550116</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
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<location offset="11567" length="4"/>
<text>Ox40</text>
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<annotation id="256">
<infon key="score">0.7644723</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11572" length="3"/>
<text>ADE</text>
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<annotation id="1188">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="11581" length="2"/>
<text>SL</text>
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<annotation id="257">
<infon key="score">0.98503137</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11602" length="3"/>
<text>CDE</text>
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<annotation id="258">
<infon key="score">0.9581188</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:37:44Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11606" length="7"/>
<text>triloop</text>
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<annotation id="1306">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:04Z</infon>
<location offset="11628" length="15"/>
<text>U-rich hexaloop</text>
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<annotation id="259">
<infon key="score">0.52676684</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11651" length="4"/>
<text>Ox40</text>
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<annotation id="260">
<infon key="score">0.64240104</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11656" length="3"/>
<text>ADE</text>
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<annotation id="1189">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="11665" length="2"/>
<text>SL</text>
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<annotation id="261">
<infon key="score">0.65158087</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="11668" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="262">
<infon key="score">0.9272881</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11693" length="7"/>
<text>surface</text>
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<annotation id="1266">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<location offset="11708" length="3"/>
<text>ROQ</text>
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<annotation id="263">
<infon key="score">0.98314255</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11750" length="3"/>
<text>CDE</text>
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<annotation id="264">
<infon key="score">0.9670553</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:07Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11754" length="7"/>
<text>triloop</text>
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<annotation id="265">
<infon key="score">0.9995376</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:10Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11860" length="6"/>
<text>Phe255</text>
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<annotation id="266">
<infon key="score">0.9970485</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="11876" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="267">
<infon key="score">0.99941254</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11925" length="3"/>
<text>U11</text>
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<annotation id="268">
<infon key="score">0.9979755</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:43Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11961" length="9"/>
<text>structure</text>
</annotation>
<annotation id="269">
<infon key="score">0.98815536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="11978" length="3"/>
<text>Tnf</text>
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<annotation id="270">
<infon key="score">0.95641446</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11982" length="3"/>
<text>CDE</text>
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<annotation id="271">
<infon key="score">0.95013833</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="11986" length="7"/>
<text>triloop</text>
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<annotation id="272">
<infon key="score">0.9995419</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="11998" length="6"/>
<text>Tyr250</text>
</annotation>
<annotation id="273">
<infon key="score">0.99712324</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:50Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12033" length="13"/>
<text>hydrogen bond</text>
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<annotation id="274">
<infon key="score">0.9995309</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:24Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12073" length="3"/>
<text>G12</text>
</annotation>
<annotation id="275">
<infon key="score">0.94750226</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12128" length="8"/>
<text>hexaloop</text>
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<annotation id="276">
<infon key="score">0.9995185</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12177" length="6"/>
<text>Tyr250</text>
</annotation>
<annotation id="277">
<infon key="score">0.9994367</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12216" length="3"/>
<text>U11</text>
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<annotation id="278">
<infon key="score">0.988433</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12286" length="21"/>
<text>stacking interactions</text>
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<annotation id="279">
<infon key="score">0.999371</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12317" length="3"/>
<text>U10</text>
</annotation>
<annotation id="280">
<infon key="score">0.99931526</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12325" length="3"/>
<text>U11</text>
</annotation>
<annotation id="281">
<infon key="score">0.9995111</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12393" length="6"/>
<text>Tyr250</text>
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<annotation id="282">
<infon key="score">0.99948704</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:46Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12435" length="3"/>
<text>U13</text>
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<annotation id="283">
<infon key="score">0.9995566</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:51Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12465" length="6"/>
<text>Val257</text>
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<annotation id="284">
<infon key="score">0.99952316</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12476" length="6"/>
<text>Lys259</text>
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<annotation id="285">
<infon key="score">0.97526985</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:01Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12486" length="6"/>
<text>strand</text>
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<annotation id="286">
<infon key="score">0.9988292</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12493" length="2"/>
<text>β3</text>
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<annotation id="287">
<infon key="score">0.63233346</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:03:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12523" length="3"/>
<text>UGU</text>
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<annotation id="288">
<infon key="score">0.862775</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:40:04Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12527" length="7"/>
<text>triloop</text>
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<annotation id="289">
<infon key="score">0.8768791</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="12542" length="3"/>
<text>Tnf</text>
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<annotation id="290">
<infon key="score">0.85423577</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12546" length="3"/>
<text>CDE</text>
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<annotation id="291">
<infon key="score">0.82655025</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12550" length="3"/>
<text>RNA</text>
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<annotation id="292">
<infon key="score">0.9614888</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12604" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="293">
<infon key="score">0.9995204</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12632" length="6"/>
<text>Lys259</text>
</annotation>
<annotation id="294">
<infon key="score">0.997125</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:12Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12645" length="14"/>
<text>hydrogen bonds</text>
</annotation>
<annotation id="295">
<infon key="score">0.99947935</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12689" length="3"/>
<text>U10</text>
</annotation>
<annotation id="296">
<infon key="score">0.99946886</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12697" length="3"/>
<text>U11</text>
</annotation>
<annotation id="297">
<infon key="score">0.9995478</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12747" length="6"/>
<text>Val257</text>
</annotation>
<annotation id="298">
<infon key="score">0.9861222</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:42:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="12754" length="6"/>
<text>stacks</text>
</annotation>
<annotation id="299">
<infon key="score">0.9994474</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12770" length="3"/>
<text>U11</text>
</annotation>
<annotation id="300">
<infon key="score">0.9847534</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="12796" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="301">
<infon key="score">0.73149467</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="12800" length="4"/>
<text>stem</text>
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<infon key="type">bond_interaction</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:31Z</infon>
<location offset="12820" length="22"/>
<text>Watson–Crick base pair</text>
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<annotation id="302">
<infon key="score">0.4196095</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="12844" length="2"/>
<text>C8</text>
</annotation>
<annotation id="303">
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<location offset="13018" length="3"/>
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<infon key="updated_at">2023-09-15T14:04:02Z</infon>
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<location offset="13112" length="3"/>
<text>A14</text>
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<location offset="13131" length="3"/>
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<location offset="13155" length="12"/>
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<infon key="updated_at">2023-09-15T12:38:43Z</infon>
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<location offset="13225" length="20"/>
<text>stacking interaction</text>
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<location offset="13249" length="2"/>
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<infon key="identifier">DUMMY:</infon>
<location offset="13253" length="3"/>
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<annotation id="319">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="13308" length="3"/>
<text>RNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13312" length="4"/>
<text>stem</text>
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<annotation id="321">
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<infon key="updated_at">2023-09-15T12:39:21Z</infon>
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<infon key="identifier">DUMMY:</infon>
<location offset="13340" length="3"/>
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<infon key="updated_at">2023-09-15T12:41:39Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13350" length="5"/>
<text>stack</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="13395" length="3"/>
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<infon key="updated_at">2023-09-15T12:41:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13406" length="4"/>
<text>wing</text>
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<annotation id="325">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:34Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="13464" length="3"/>
<text>C12</text>
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<annotation id="1309">
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<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:06Z</infon>
<location offset="13475" length="5"/>
<text>Ox-40</text>
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<annotation id="1111">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="13481" length="3"/>
<text>ADE</text>
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<annotation id="1191">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="13490" length="2"/>
<text>SL</text>
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<annotation id="1074">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="13602" length="3"/>
<text>CDE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="13606" length="2"/>
<text>SL</text>
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<annotation id="1112">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="13619" length="3"/>
<text>ADE</text>
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<annotation id="1193">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="13623" length="2"/>
<text>SL</text>
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<annotation id="1113">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="13630" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="13639" length="2"/>
<text>SL</text>
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<annotation id="1279">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="13642" length="4"/>
<text>RNAs</text>
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<annotation id="1269">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<location offset="13762" length="3"/>
<text>ROQ</text>
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<annotation id="1242">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="13836" length="3"/>
<text>RNA</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
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<text>NMR analysis of ROQ interactions with ADE SLs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:00Z</infon>
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<location offset="13852" length="3"/>
<text>NMR</text>
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<annotation id="327">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
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<location offset="13868" length="3"/>
<text>ROQ</text>
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<annotation id="328">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<location offset="13890" length="3"/>
<text>ADE</text>
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<annotation id="329">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="13894" length="3"/>
<text>SLs</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>13898</offset>
<text>We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).</text>
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<infon key="score">0.998158</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:42:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="13911" length="16"/>
<text>NMR spectroscopy</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
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<location offset="13943" length="3"/>
<text>ROQ</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="13969" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="13982" length="3"/>
<text>CDE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="13991" length="2"/>
<text>SL</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="13994" length="4"/>
<text>RNAs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:31:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14012" length="4"/>
<text>CSPs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14044" length="3"/>
<text>ROQ</text>
</annotation>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14073" length="4"/>
<text>Ox40</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="14078" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="14087" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="14090" length="3"/>
<text>RNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14162" length="3"/>
<text>CDE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14166" length="3"/>
<text>SLs</text>
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<annotation id="337">
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<infon key="updated_at">2023-09-15T12:42:55Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14179" length="6"/>
<text>Lys220</text>
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<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14187" length="6"/>
<text>Lys239</text>
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<infon key="updated_at">2023-09-15T12:43:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14194" length="6"/>
<text>Thr240</text>
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<infon key="updated_at">2023-09-15T12:39:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14205" length="6"/>
<text>Lys259</text>
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<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14212" length="6"/>
<text>Arg260</text>
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<infon key="score">0.9986762</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14293" length="17"/>
<text>crystal structure</text>
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<annotation id="343">
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:47Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14361" length="15"/>
<text>binding surface</text>
</annotation>
<annotation id="344">
<infon key="score">0.9667672</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:19Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14410" length="15"/>
<text>CSP differences</text>
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<annotation id="345">
<infon key="score">0.9985941</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14456" length="3"/>
<text>ROQ</text>
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<annotation id="346">
<infon key="score">0.860187</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14470" length="4"/>
<text>Ox40</text>
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<annotation id="1116">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="14475" length="3"/>
<text>ADE</text>
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<annotation id="1197">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="14484" length="2"/>
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<annotation id="1281">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="14487" length="4"/>
<text>RNAs</text>
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<annotation id="1076">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="14503" length="3"/>
<text>CDE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<location offset="14512" length="2"/>
<text>SL</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="14515" length="3"/>
<text>RNA</text>
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<annotation id="347">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14526" length="4"/>
<text>Ox40</text>
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<annotation id="348">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14531" length="6"/>
<text>3′-UTR</text>
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<annotation id="349">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14559" length="3"/>
<text>Tnf</text>
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<annotation id="350">
<infon key="score">0.40692672</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14563" length="3"/>
<text>CDE</text>
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<annotation id="1199">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="14567" length="2"/>
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<annotation id="1245">
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="14570" length="3"/>
<text>RNA</text>
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<annotation id="351">
<infon key="score">0.9994935</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:32:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14635" length="6"/>
<text>Ser253</text>
</annotation>
<annotation id="352">
<infon key="score">0.96339715</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="14686" length="4"/>
<text>Ox40</text>
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<annotation id="1117">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="14691" length="3"/>
<text>ADE</text>
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<annotation id="1200">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="14700" length="2"/>
<text>SL</text>
</annotation>
<annotation id="353">
<infon key="score">0.99272835</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:58Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14756" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="354">
<infon key="score">0.99858105</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14809" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="355">
<infon key="score">0.8455614</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="14837" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1118">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="14854" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1201">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="14863" length="2"/>
<text>SL</text>
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<annotation id="1282">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="14866" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="356">
<infon key="score">0.99870276</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="14898" length="3"/>
<text>NMR</text>
</annotation>
<annotation id="357">
<infon key="score">0.98940325</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:30:44Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14902" length="7"/>
<text>spectra</text>
</annotation>
<annotation id="358">
<infon key="score">0.71015626</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:31:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="14914" length="4"/>
<text>CSPs</text>
</annotation>
<annotation id="1246">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="14993" length="3"/>
<text>RNA</text>
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<annotation id="359">
<infon key="score">0.9961659</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15016" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="360">
<infon key="score">0.9940701</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="15038" length="4"/>
<text>RNAs</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">title_2</infon>
<offset>15076</offset>
<text>Mutational analysis of the ROQ-ADE interaction</text>
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<infon key="score">0.99872553</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15076" length="19"/>
<text>Mutational analysis</text>
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<annotation id="1270">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<location offset="15103" length="3"/>
<text>ROQ</text>
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<annotation id="1119">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="15107" length="3"/>
<text>ADE</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>15123</offset>
<text>To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.</text>
<annotation id="362">
<infon key="score">0.57086194</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15166" length="3"/>
<text>ROQ</text>
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<annotation id="363">
<infon key="score">0.9989199</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:28Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15228" length="37"/>
<text>electrophoretic mobility shift assays</text>
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<annotation id="364">
<infon key="score">0.99855083</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15267" length="5"/>
<text>EMSAs</text>
</annotation>
<annotation id="365">
<infon key="score">0.9794989</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15295" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="366">
<infon key="score">0.99387276</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15314" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1120">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="15319" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1247">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="15328" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="367">
<infon key="score">0.99898165</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15401" length="9"/>
<text>wild-type</text>
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<annotation id="368">
<infon key="score">0.9987429</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15411" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="369">
<infon key="score">0.9966923</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15436" length="8"/>
<text>affinity</text>
</annotation>
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<infon key="score">0.9985247</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="15475" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="371">
<infon key="score">0.9801728</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15479" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="372">
<infon key="score">0.9979738</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15618" length="17"/>
<text>crystal structure</text>
</annotation>
<annotation id="373">
<infon key="score">0.99468035</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15653" length="3"/>
<text>NMR</text>
</annotation>
<annotation id="374">
<infon key="score">0.97366476</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:31:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15657" length="4"/>
<text>CSPs</text>
</annotation>
<annotation id="1330">
<infon key="type">complex_assembly</infon>
<infon key="identifier">GO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:39Z</infon>
<location offset="15706" length="11"/>
<text>ROQ-Tnf CDE</text>
</annotation>
<annotation id="375">
<infon key="score">0.995828</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15784" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="376">
<infon key="score">0.9995509</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:42:56Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15822" length="6"/>
<text>Lys220</text>
</annotation>
<annotation id="377">
<infon key="score">0.99954456</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:01Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15830" length="6"/>
<text>Lys239</text>
</annotation>
<annotation id="378">
<infon key="score">0.99953663</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15838" length="6"/>
<text>Lys259</text>
</annotation>
<annotation id="379">
<infon key="score">0.9995377</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:43:13Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15849" length="6"/>
<text>Arg260</text>
</annotation>
<annotation id="380">
<infon key="score">0.99847454</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:45Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15897" length="11"/>
<text>replacement</text>
</annotation>
<annotation id="381">
<infon key="score">0.9950722</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15912" length="7"/>
<text>alanine</text>
</annotation>
<annotation id="382">
<infon key="score">0.99901664</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="15979" length="5"/>
<text>Y250A</text>
</annotation>
<annotation id="383">
<infon key="score">0.9990872</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="15985" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="384">
<infon key="score">0.819331</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="15999" length="8"/>
<text>hexaloop</text>
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<annotation id="1202">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="16008" length="2"/>
<text>SL</text>
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<annotation id="1248">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="16011" length="3"/>
<text>RNA</text>
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<annotation id="385">
<infon key="score">0.99883825</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16048" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="386">
<infon key="score">0.99365145</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16052" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="387">
<infon key="score">0.9995796</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16114" length="6"/>
<text>Tyr250</text>
</annotation>
<annotation id="388">
<infon key="score">0.8907902</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16146" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="389">
<infon key="score">0.9959369</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16184" length="21"/>
<text>stacking interactions</text>
</annotation>
<annotation id="390">
<infon key="score">0.9981415</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:06Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16219" length="8"/>
<text>Mutation</text>
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<annotation id="391">
<infon key="score">0.99959916</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:32:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16231" length="6"/>
<text>Ser253</text>
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<annotation id="392">
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<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:31:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16257" length="4"/>
<text>CSPs</text>
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<annotation id="1352">
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<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:59:07Z</infon>
<location offset="16269" length="14"/>
<text>NMR titrations</text>
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<annotation id="393">
<infon key="score">0.98001057</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16379" length="21"/>
<text>chemical shift change</text>
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<annotation id="394">
<infon key="score">0.9994816</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16482" length="3"/>
<text>U11</text>
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<annotation id="395">
<infon key="score">0.999371</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16490" length="3"/>
<text>U13</text>
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<annotation id="396">
<infon key="score">0.99785703</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16512" length="6"/>
<text>mutant</text>
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<annotation id="397">
<infon key="score">0.9994467</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16526" length="4"/>
<text>wing</text>
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<annotation id="398">
<infon key="score">0.9115498</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16538" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="399">
<infon key="score">0.99896014</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16550" length="5"/>
<text>S265Y</text>
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<annotation id="400">
<infon key="score">0.9987878</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="16653" length="3"/>
<text>Tnf</text>
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<annotation id="401">
<infon key="score">0.99510777</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16657" length="3"/>
<text>CDE</text>
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<annotation id="402">
<infon key="score">0.99833244</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16705" length="11"/>
<text>replacement</text>
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<annotation id="403">
<infon key="score">0.99740535</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:45:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16720" length="3"/>
<text>Tyr</text>
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<annotation id="404">
<infon key="score">0.8353558</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="16753" length="3"/>
<text>RNA</text>
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<annotation id="405">
<infon key="score">0.9951656</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:49Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="16835" length="19"/>
<text>mutational analysis</text>
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<annotation id="406">
<infon key="score">0.8165691</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:46:05Z</infon>
<infon key="identifier">SO:</infon>
<location offset="16903" length="8"/>
<text>hexaloop</text>
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<annotation id="407">
<infon key="score">0.99865377</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:59:12Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16928" length="18"/>
<text>crystal structures</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>16950</offset>
<text>To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). The observed partial rescue may relate to the low, close to endogenous expression of these constructs (Supplementary Fig. 5b). However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.</text>
<annotation id="408">
<infon key="score">0.99958366</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="16995" length="6"/>
<text>Tyr250</text>
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<annotation id="409">
<infon key="score">0.998746</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17005" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="410">
<infon key="score">0.9991165</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17017" length="4"/>
<text>Ox40</text>
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<annotation id="411">
<infon key="score">0.9979837</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17022" length="4"/>
<text>mRNA</text>
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<infon key="score">0.99830437</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17067" length="32"/>
<text>retroviral reconstitution system</text>
</annotation>
<annotation id="413">
<infon key="score">0.2854319</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17103" length="6"/>
<text>Roquin</text>
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<annotation id="1331">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:38:23Z</infon>
<location offset="17161" length="5"/>
<text>Rc3h1</text>
</annotation>
<annotation id="1334">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:39:25Z</infon>
<location offset="17167" length="3"/>
<text>2fl</text>
</annotation>
<annotation id="1335">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:39:40Z</infon>
<location offset="17171" length="2"/>
<text>fl</text>
</annotation>
<annotation id="1154">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:08Z</infon>
<location offset="17194" length="4"/>
<text>mice</text>
</annotation>
<annotation id="414">
<infon key="score">0.8110845</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17217" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="415">
<infon key="score">0.46404725</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:48:33Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17226" length="1"/>
<text>2</text>
</annotation>
<annotation id="1311">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:01Z</infon>
<location offset="17248" length="9"/>
<text>tamoxifen</text>
</annotation>
<annotation id="416">
<infon key="score">0.87689686</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:55:03Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17292" length="46"/>
<text>reverse tetracycline-controlled transactivator</text>
</annotation>
<annotation id="417">
<infon key="score">0.96973455</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:00Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17339" length="4"/>
<text>rtTA</text>
</annotation>
<annotation id="418">
<infon key="score">0.9987544</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:18Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17371" length="19"/>
<text>4-hydroxy tamoxifen</text>
</annotation>
<annotation id="419">
<infon key="score">0.49092653</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17448" length="11"/>
<text>doxycycline</text>
</annotation>
<annotation id="420">
<infon key="score">0.9383189</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17505" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="421">
<infon key="score">0.8359207</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17549" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="422">
<infon key="score">0.93609476</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:07Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17568" length="9"/>
<text>tamoxifen</text>
</annotation>
<annotation id="423">
<infon key="score">0.99917966</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17653" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="424">
<infon key="score">0.9992281</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17662" length="4"/>
<text>Icos</text>
</annotation>
<annotation id="425">
<infon key="score">0.9139179</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:13:35Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17722" length="23"/>
<text>costimulatory receptors</text>
</annotation>
<annotation id="426">
<infon key="score">0.910425</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:22Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="17777" length="11"/>
<text>doxycycline</text>
</annotation>
<annotation id="427">
<infon key="score">0.95158434</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17817" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="428">
<infon key="score">0.99925894</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:46:59Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17826" length="2"/>
<text>WT</text>
</annotation>
<annotation id="429">
<infon key="score">0.9990452</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17917" length="5"/>
<text>Y250A</text>
</annotation>
<annotation id="430">
<infon key="score">0.999201</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17923" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="431">
<infon key="score">0.99857616</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="17933" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="432">
<infon key="score">0.99902654</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:26Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17949" length="5"/>
<text>K220A</text>
</annotation>
<annotation id="433">
<infon key="score">0.99901557</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:31Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17956" length="5"/>
<text>K239A</text>
</annotation>
<annotation id="434">
<infon key="score">0.9989231</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:36Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="17966" length="4"/>
<text>R260</text>
</annotation>
<annotation id="435">
<infon key="score">0.99922574</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:04Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="17971" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="436">
<infon key="score">0.711355</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18009" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="437">
<infon key="score">0.65221393</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18013" length="2"/>
<text>SL</text>
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<annotation id="1310">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:32Z</infon>
<location offset="18236" length="14"/>
<text>overexpression</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:12Z</infon>
<infon key="identifier">PR:</infon>
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<text>Icos</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18471" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:11Z</infon>
<infon key="identifier">MESH:</infon>
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<text>structure–function analyses</text>
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<infon key="updated_at">2023-09-15T12:50:18Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>Y250</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
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<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
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<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
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<text>Roquin</text>
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<annotation id="446">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:12Z</infon>
<infon key="identifier">PR:</infon>
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<text>Icos</text>
</annotation>
</passage>
<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>18696</offset>
<text>We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="18759" length="4"/>
<text>Ox40</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="18764" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="18773" length="2"/>
<text>SL</text>
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<annotation id="448">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="18807" length="3"/>
<text>ROQ</text>
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<annotation id="449">
<infon key="score">0.9972644</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="18960" length="20"/>
<text>co-crystal structure</text>
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<annotation id="450">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:11:00Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19019" length="3"/>
<text>NMR</text>
</annotation>
<annotation id="451">
<infon key="score">0.99574333</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19051" length="6"/>
<text>mutant</text>
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<infon key="score">0.9964078</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19058" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="453">
<infon key="score">0.9985248</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:37Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19097" length="11"/>
<text>stem region</text>
</annotation>
<annotation id="454">
<infon key="score">0.99884385</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19127" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1314">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:55:31Z</infon>
<location offset="19137" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1204">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="19146" length="2"/>
<text>SL</text>
</annotation>
<annotation id="455">
<infon key="score">0.99892396</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19198" length="25"/>
<text>surface plasmon resonance</text>
</annotation>
<annotation id="456">
<infon key="score">0.9980252</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:54:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19249" length="22"/>
<text>dissociation constants</text>
</annotation>
<annotation id="457">
<infon key="score">0.9205294</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19280" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="458">
<infon key="score">0.98794657</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="19284" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="459">
<infon key="score">0.99840325</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:15Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19349" length="11"/>
<text>replacement</text>
</annotation>
<annotation id="460">
<infon key="score">0.81623113</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:50Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19366" length="2"/>
<text>C8</text>
</annotation>
<annotation id="461">
<infon key="score">0.9292495</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19369" length="3"/>
<text>G15</text>
</annotation>
<annotation id="1312">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:54:39Z</infon>
<location offset="19394" length="1"/>
<text>A</text>
</annotation>
<annotation id="1313">
<infon key="type">residue_name</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:54:54Z</infon>
<location offset="19396" length="1"/>
<text>U</text>
</annotation>
<annotation id="462">
<infon key="score">0.99763864</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19399" length="5"/>
<text>Mut 4</text>
</annotation>
<annotation id="463">
<infon key="score">0.9982413</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:55:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19423" length="8"/>
<text>affinity</text>
</annotation>
<annotation id="464">
<infon key="score">0.9934295</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:55:03Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19456" length="4"/>
<text>loop</text>
</annotation>
<annotation id="465">
<infon key="score">0.9989876</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19473" length="4"/>
<text>A14C</text>
</annotation>
<annotation id="466">
<infon key="score">0.99875975</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19478" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="467">
<infon key="score">0.9978521</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:44Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19486" length="5"/>
<text>Mut 1</text>
</annotation>
<annotation id="468">
<infon key="score">0.99799025</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:59:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19518" length="8"/>
<text>affinity</text>
</annotation>
<annotation id="469">
<infon key="score">0.9988158</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:19Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19558" length="25"/>
<text>surface plasmon resonance</text>
</annotation>
<annotation id="470">
<infon key="score">0.9981153</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:41Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19611" length="5"/>
<text>Mut 1</text>
</annotation>
<annotation id="471">
<infon key="score">0.9308423</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:53:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19702" length="9"/>
<text>tetraloop</text>
</annotation>
<annotation id="472">
<infon key="score">0.95997465</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:53:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19723" length="1"/>
<text>G</text>
</annotation>
<annotation id="473">
<infon key="score">0.5003149</infon>
<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:53:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19725" length="1"/>
<text>C</text>
</annotation>
<annotation id="474">
<infon key="score">0.998454</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:56:23Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19790" length="19"/>
<text>structural analysis</text>
</annotation>
<annotation id="475">
<infon key="score">0.98113084</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:53:27Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19850" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="476">
<infon key="score">0.9990087</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="19910" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="477">
<infon key="score">0.9951514</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:38:38Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19929" length="21"/>
<text>stacking interactions</text>
</annotation>
<annotation id="478">
<infon key="score">0.9995321</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:41:45Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19959" length="3"/>
<text>G15</text>
</annotation>
<annotation id="479">
<infon key="score">0.9995315</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:30Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19964" length="2"/>
<text>G9</text>
</annotation>
<annotation id="480">
<infon key="score">0.9995627</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:18Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19971" length="4"/>
<text>Y250</text>
</annotation>
<annotation id="481">
<infon key="score">0.999084</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:52Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19983" length="3"/>
<text>G9C</text>
</annotation>
<annotation id="482">
<infon key="score">0.9987691</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="19987" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="483">
<infon key="score">0.998194</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:56Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="19995" length="5"/>
<text>Mut 2</text>
</annotation>
<annotation id="484">
<infon key="score">0.99908125</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20034" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="485">
<infon key="score">0.7375572</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20045" length="2"/>
<text>SL</text>
</annotation>
<annotation id="486">
<infon key="score">0.9985176</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="20048" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="487">
<infon key="score">0.9990637</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:59Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20124" length="8"/>
<text>U11AU13G</text>
</annotation>
<annotation id="488">
<infon key="score">0.87387824</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:53:20Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20133" length="13"/>
<text>double mutant</text>
</annotation>
<annotation id="489">
<infon key="score">0.99743533</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:08:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="20148" length="5"/>
<text>Mut 3</text>
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<infon key="updated_at">2023-09-15T12:57:01Z</infon>
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<infon key="updated_at">2023-09-15T12:21:53Z</infon>
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<text>SELEX</text>
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<text>loop</text>
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<text>affinity</text>
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<infon key="updated_at">2023-09-15T12:56:47Z</infon>
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<text>wild-type</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
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<text>Tnf</text>
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<infon key="annotator">cleaner0</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
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<text>Ox40</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
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<text>SLs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20527" length="3"/>
<text>ROQ</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
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<text>Roquin binding to different SLs in the Ox40 3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:56Z</infon>
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<text>SLs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20659" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20664" length="6"/>
<text>3′-UTR</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
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<text>We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.</text>
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<text>Roquin-1</text>
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<text>CDE</text>
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<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20743" length="6"/>
<text>3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20753" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>mRNA</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="20841" length="3"/>
<text>CDE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="20858" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="20867" length="2"/>
<text>SL</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="20870" length="4"/>
<text>RNAs</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20894" length="8"/>
<text>Roquin-1</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20933" length="11"/>
<text>full-length</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="20945" length="4"/>
<text>Ox40</text>
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<infon key="score">0.99523497</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="20950" length="6"/>
<text>3′-UTR</text>
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<infon key="score">0.99872977</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:57:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="20962" length="18"/>
<text>binding affinities</text>
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<infon key="score">0.9112169</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21005" length="17"/>
<text>N-terminal domain</text>
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<annotation id="520">
<infon key="score">0.9990762</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21026" length="8"/>
<text>Roquin-1</text>
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<infon key="score">0.99726003</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:57:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21045" length="5"/>
<text>2–440</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21085" length="3"/>
<text>ROQ</text>
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<infon key="score">0.7369649</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21096" length="5"/>
<text>alone</text>
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<infon key="score">0.9983428</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:57:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="21140" length="22"/>
<text>dissociation constants</text>
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<infon key="score">0.99771035</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21171" length="3"/>
<text>ROQ</text>
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<infon key="score">0.7745781</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21196" length="4"/>
<text>Ox40</text>
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<annotation id="1079">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="21201" length="3"/>
<text>CDE</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="21210" length="2"/>
<text>SL</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="21221" length="3"/>
<text>ADE</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="21230" length="2"/>
<text>SL</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="21233" length="4"/>
<text>RNAs</text>
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<infon key="score">0.99896085</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:57:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21320" length="17"/>
<text>binding interface</text>
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<infon key="score">0.97753364</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:55Z</infon>
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<location offset="21384" length="8"/>
<text>hexaloop</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:50:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21437" length="8"/>
<text>hexaloop</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21446" length="2"/>
<text>SL</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>RNA</text>
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<infon key="score">0.687771</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="21460" length="4"/>
<text>Ox40</text>
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<infon key="score">0.9913631</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="21465" length="6"/>
<text>3′-UTR</text>
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<infon key="score">0.9859484</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
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<text>3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:09Z</infon>
<infon key="identifier">MESH:</infon>
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<text>point mutagenesis</text>
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<infon key="updated_at">2023-09-15T13:51:03Z</infon>
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<location offset="21570" length="11"/>
<text>stem region</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="21615" length="2"/>
<text>SL</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="21618" length="3"/>
<text>RNA</text>
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<text>2–440</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:31Z</infon>
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<text>Gel shift assays</text>
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<infon key="identifier">DUMMY:</infon>
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<location offset="21882" length="10"/>
<text>titrations</text>
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<infon key="identifier">CHEBI:</infon>
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<location offset="21939" length="3"/>
<text>RNA</text>
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<text>SL</text>
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<text>RNA</text>
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<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22213" length="6"/>
<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:48Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>wild-type</text>
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<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<infon key="identifier">SO:</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22310" length="6"/>
<text>Roquin</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:14Z</infon>
<location offset="22341" length="2"/>
<text>SL</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="22344" length="3"/>
<text>RNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22377" length="11"/>
<text>full-length</text>
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<annotation id="552">
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<infon key="updated_at">2023-09-15T12:07:57Z</infon>
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<location offset="22389" length="4"/>
<text>Ox40</text>
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<infon key="identifier">SO:</infon>
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<text>3′-UTR</text>
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</passage>
<passage>
<infon key="section_type">RESULTS</infon>
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<text>Regulation of Ox40 expression via two motifs in its 3′-UTR</text>
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<location offset="22434" length="4"/>
<text>Ox40</text>
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<annotation id="555">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
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<text>3′-UTR</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
<offset>22481</offset>
<text>To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).</text>
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<infon key="identifier">SO:</infon>
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<text>ADE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:31Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>mRNA</text>
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<infon key="updated_at">2023-09-15T13:56:52Z</infon>
<infon key="identifier">MESH:</infon>
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<text>introduced</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22572" length="4"/>
<text>Ox40</text>
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<infon key="score">0.9963111</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>mRNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:08Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22602" length="7"/>
<text>altered</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22610" length="7"/>
<text>3′-UTRs</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="22667" length="3"/>
<text>ADE</text>
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<annotation id="1080">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="22680" length="3"/>
<text>CDE</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="22689" length="2"/>
<text>SL</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="22692" length="4"/>
<text>RNAs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:55Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22704" length="6"/>
<text>3′-UTR</text>
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<infon key="score">0.9983773</infon>
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<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22745" length="6"/>
<text>Roquin</text>
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<infon key="score">0.99746215</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="22795" length="4"/>
<text>Ox40</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="22884" length="3"/>
<text>RNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:01:09Z</infon>
<infon key="identifier">MESH:</infon>
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<text>deletion and point mutagenesis</text>
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<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="22931" length="10"/>
<text>abrogating</text>
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<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:01:02Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="22942" length="12"/>
<text>base pairing</text>
</annotation>
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<infon key="score">0.9992924</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:51:08Z</infon>
<infon key="identifier">SO:</infon>
<location offset="22962" length="11"/>
<text>stem region</text>
</annotation>
<annotation id="569">
<infon key="score">0.9529885</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:57Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23033" length="12"/>
<text>retroviruses</text>
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<infon key="score">0.9859139</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23060" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="571">
<infon key="score">0.9985753</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="23125" length="7"/>
<text>3′-UTRs</text>
</annotation>
<annotation id="1061">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<location offset="23138" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="1318">
<infon key="type">protein</infon>
<infon key="identifier">PR:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:00:57Z</infon>
<location offset="23147" length="1"/>
<text>2</text>
</annotation>
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<infon key="score">0.9797829</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23159" length="5"/>
<text>mouse</text>
</annotation>
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<infon key="score">0.9972741</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23188" length="11"/>
<text>Doxycycline</text>
</annotation>
<annotation id="574">
<infon key="score">0.99609536</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23255" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="575">
<infon key="score">0.9766459</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:00:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23350" length="10"/>
<text>lentiviral</text>
</annotation>
<annotation id="576">
<infon key="score">0.99310863</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23411" length="4"/>
<text>Ox40</text>
</annotation>
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<infon key="score">0.99667835</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23442" length="11"/>
<text>doxycycline</text>
</annotation>
<annotation id="578">
<infon key="score">0.9986136</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:57:07Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="23487" length="14"/>
<text>flow cytometry</text>
</annotation>
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<infon key="score">0.99719197</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23550" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="580">
<infon key="score">0.90989846</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:01:22Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23555" length="29"/>
<text>mean fluorescence intensities</text>
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<infon key="score">0.9950245</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="23611" length="11"/>
<text>doxycycline</text>
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<infon key="score">0.9867207</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="23684" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="583">
<infon key="score">0.9982474</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:27Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23700" length="7"/>
<text>without</text>
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<text>3′-UTR</text>
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<infon key="updated_at">2023-09-15T13:01:31Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="23898" length="4"/>
<text>1–80</text>
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<text>double mut</text>
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<text>mutations</text>
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<infon key="updated_at">2023-09-15T13:01:24Z</infon>
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<location offset="24016" length="8"/>
<text>hexaloop</text>
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<text>3′-UTR</text>
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<text>1–120</text>
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<location offset="24042" length="7"/>
<text>CDE mut</text>
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<annotation id="1081">
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<infon key="updated_at">2023-09-15T12:08:48Z</infon>
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<text>CDE</text>
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<text>triloop</text>
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<text>intact</text>
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<infon key="updated_at">2023-09-15T12:06:56Z</infon>
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<text>Roquin</text>
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<text>3′-UTR</text>
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<passage>
<infon key="section_type">RESULTS</infon>
<infon key="type">paragraph</infon>
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<text>To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.</text>
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<text>Roquin</text>
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<text>3′-UTR</text>
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<text>Ox40</text>
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<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>doxycycline</text>
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<infon key="updated_at">2023-09-15T13:02:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24531" length="38"/>
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<infon key="updated_at">2023-09-15T12:07:58Z</infon>
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<text>Ox40</text>
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<infon key="updated_at">2023-09-15T12:59:32Z</infon>
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<infon key="updated_at">2023-09-15T12:16:54Z</infon>
<infon key="identifier">MESH:</infon>
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<infon key="updated_at">2023-09-15T12:09:55Z</infon>
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<text>3′-UTR</text>
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<infon key="updated_at">2023-09-15T13:02:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24646" length="3"/>
<text>CDS</text>
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<infon key="score">0.9904898</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24650" length="4"/>
<text>t1/2</text>
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<infon key="updated_at">2023-09-15T12:58:48Z</infon>
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<location offset="24666" length="11"/>
<text>full-length</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24678" length="4"/>
<text>t1/2</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:37Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24735" length="17"/>
<text>combined mutation</text>
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<location offset="24760" length="3"/>
<text>CDE</text>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="24777" length="3"/>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="24786" length="3"/>
<text>SLs</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="24791" length="3"/>
<text>ADE</text>
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<annotation id="1083">
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="24795" length="3"/>
<text>CDE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:04:38Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24804" length="3"/>
<text>mut</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24808" length="4"/>
<text>t1/2</text>
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<infon key="updated_at">2023-09-15T13:57:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="24834" length="9"/>
<text>mutations</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="24858" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="24874" length="3"/>
<text>CDE</text>
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<infon key="updated_at">2023-09-15T12:58:56Z</infon>
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<location offset="24883" length="3"/>
<text>SLs</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="24916" length="3"/>
<text>ADE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:04:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24925" length="3"/>
<text>mut</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:15Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24929" length="4"/>
<text>t1/2</text>
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<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-15T12:08:48Z</infon>
<location offset="24943" length="3"/>
<text>CDE</text>
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<infon key="updated_at">2023-09-15T13:04:39Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="24952" length="3"/>
<text>mut</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:03:15Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="25118" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<text>mRNA</text>
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<passage>
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<text>Discussion</text>
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<passage>
<infon key="section_type">DISCUSS</infon>
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<text>Recent structural and functional studies have provided first insight into the RNA binding of Roquin. Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. Some evidence for this is provided by a recent study by Landthaler and colleagues.</text>
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<infon key="updated_at">2023-09-15T13:04:50Z</infon>
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<infon key="updated_at">2023-09-15T12:06:56Z</infon>
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<text>Roquin</text>
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<infon key="updated_at">2023-09-15T13:04:54Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
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<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-15T12:08:48Z</infon>
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<text>CDE</text>
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<text>SL</text>
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<text>Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.</text>
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<text>3′-UTRs</text>
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<infon key="updated_at">2023-09-15T12:10:46Z</infon>
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<text>SELEX</text>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<text>ADE</text>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<text>ADE</text>
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<infon key="updated_at">2023-09-15T12:07:58Z</infon>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26122" length="3"/>
<text>ADE</text>
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<infon key="identifier">SO:</infon>
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<infon key="updated_at">2023-09-15T12:17:15Z</infon>
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<infon key="identifier">CHEBI:</infon>
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<infon key="updated_at">2023-09-15T12:23:25Z</infon>
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<text>RNAs</text>
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<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="26145" length="2"/>
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<infon key="updated_at">2023-09-15T13:07:36Z</infon>
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<location offset="26161" length="8"/>
<text>hexaloop</text>
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<infon key="updated_at">2023-09-15T13:51:30Z</infon>
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<location offset="26183" length="7"/>
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<infon key="updated_at">2023-09-15T13:06:12Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26235" length="24"/>
<text>RNA-helical stem regions</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26267" length="3"/>
<text>ROQ</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:06:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26299" length="7"/>
<text>triloop</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:06:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="26311" length="8"/>
<text>hexaloop</text>
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<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:06:37Z</infon>
<location offset="26341" length="16"/>
<text>U-rich hexaloops</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<location offset="26365" length="3"/>
<text>ADE</text>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<text>ADE</text>
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<location offset="26382" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="26385" length="4"/>
<text>RNAs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:24Z</infon>
<infon key="identifier">SO:</infon>
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<text>helix</text>
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<infon key="updated_at">2023-09-15T12:36:29Z</infon>
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<location offset="26452" length="6"/>
<text>strand</text>
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<infon key="updated_at">2023-09-15T12:36:35Z</infon>
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<infon key="updated_at">2023-09-15T13:06:56Z</infon>
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<infon key="updated_at">2023-09-15T13:07:20Z</infon>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
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<infon key="updated_at">2023-09-15T13:07:14Z</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
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<infon key="updated_at">2023-09-15T12:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
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<infon key="annotator">cleaner0</infon>
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<infon key="identifier">PR:</infon>
<location offset="27000" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
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<location offset="27005" length="3"/>
<text>ADE</text>
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<infon key="annotator">cleaner0</infon>
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<infon key="identifier">SO:</infon>
<location offset="27014" length="2"/>
<text>SL</text>
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<annotation id="688">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27048" length="3"/>
<text>ROQ</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:57:24Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27075" length="11"/>
<text>replacement</text>
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<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27090" length="3"/>
<text>U11</text>
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<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27098" length="3"/>
<text>U13</text>
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<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="27181" length="3"/>
<text>RNA</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27218" length="6"/>
<text>Roquin</text>
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<annotation id="693">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27250" length="3"/>
<text>ADE</text>
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<annotation id="1087">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<location offset="27258" length="3"/>
<text>CDE</text>
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<annotation id="1289">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="27267" length="4"/>
<text>RNAs</text>
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<annotation id="694">
<infon key="score">0.8661621</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27337" length="3"/>
<text>RNA</text>
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<annotation id="695">
<infon key="score">0.7980392</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:57Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27341" length="4"/>
<text>stem</text>
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<annotation id="1325">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:08:28Z</infon>
<location offset="27382" length="18"/>
<text>hexa- and triloops</text>
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<annotation id="696">
<infon key="score">0.9992067</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27440" length="5"/>
<text>bound</text>
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<annotation id="697">
<infon key="score">0.97536874</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="27446" length="3"/>
<text>RNA</text>
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<annotation id="698">
<infon key="score">0.9976661</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:49Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27450" length="9"/>
<text>structure</text>
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<annotation id="699">
<infon key="score">0.92127097</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27476" length="18"/>
<text>tri- and hexaloops</text>
</annotation>
<annotation id="700">
<infon key="score">0.97104734</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:54Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27503" length="8"/>
<text>stacking</text>
</annotation>
<annotation id="701">
<infon key="score">0.9913735</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="27578" length="15"/>
<text>structural data</text>
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<annotation id="702">
<infon key="score">0.9992354</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27648" length="6"/>
<text>Roquin</text>
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<annotation id="1216">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="27680" length="2"/>
<text>SL</text>
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<annotation id="1255">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="27683" length="3"/>
<text>RNA</text>
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<annotation id="703">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:08:34Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27719" length="5"/>
<text>loops</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>27734</offset>
<text>Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.</text>
<annotation id="704">
<infon key="score">0.9982291</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:10:46Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27753" length="5"/>
<text>SELEX</text>
</annotation>
<annotation id="705">
<infon key="score">0.9992132</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:08:51Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27787" length="13"/>
<text>U-rich motifs</text>
</annotation>
<annotation id="706">
<infon key="score">0.9991099</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:08:53Z</infon>
<infon key="identifier">SO:</infon>
<location offset="27875" length="12"/>
<text>U-rich loops</text>
</annotation>
<annotation id="707">
<infon key="score">0.99874634</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:09:01Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27924" length="36"/>
<text>crosslinking and immunoprecipitation</text>
</annotation>
<annotation id="708">
<infon key="score">0.9992239</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="27964" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="709">
<infon key="score">0.99885863</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:09:04Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="27979" length="8"/>
<text>PAR-CLIP</text>
</annotation>
<annotation id="710">
<infon key="score">0.9274671</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:09:09Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28040" length="15"/>
<text>U-rich hexaloop</text>
</annotation>
<annotation id="711">
<infon key="score">0.9991829</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28200" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="712">
<infon key="score">0.93437153</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:09:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28220" length="16"/>
<text>structural study</text>
</annotation>
<annotation id="713">
<infon key="score">0.9968534</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28322" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="714">
<infon key="score">0.99820507</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28339" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="715">
<infon key="score">0.99075</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28352" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="716">
<infon key="score">0.9986799</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28356" length="7"/>
<text>3′-UTRs</text>
</annotation>
<annotation id="1217">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="28461" length="2"/>
<text>SL</text>
</annotation>
<annotation id="717">
<infon key="score">0.9981084</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28475" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="718">
<infon key="score">0.9981975</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:54:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28485" length="3"/>
<text>A20</text>
</annotation>
<annotation id="719">
<infon key="score">0.99777573</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28489" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="720">
<infon key="score">0.99930227</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28523" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="721">
<infon key="score">0.9984925</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28592" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="722">
<infon key="score">0.9898441</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:09:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28596" length="7"/>
<text>triloop</text>
</annotation>
<annotation id="723">
<infon key="score">0.99866307</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:54:13Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28690" length="3"/>
<text>A20</text>
</annotation>
<annotation id="724">
<infon key="score">0.9991019</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28694" length="2"/>
<text>SL</text>
</annotation>
<annotation id="725">
<infon key="score">0.999321</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28700" length="6"/>
<text>Roquin</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>28712</offset>
<text>The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).</text>
<annotation id="726">
<infon key="score">0.9979055</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:53:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28727" length="16"/>
<text>cis RNA elements</text>
</annotation>
<annotation id="727">
<infon key="score">0.9985352</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="28747" length="7"/>
<text>3′-UTRs</text>
</annotation>
<annotation id="728">
<infon key="score">0.9989159</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="28844" length="12"/>
<text>endonuclease</text>
</annotation>
<annotation id="729">
<infon key="score">0.9992896</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:21Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28857" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="730">
<infon key="score">0.99783343</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="28884" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="731">
<infon key="score">0.9970878</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="28941" length="5"/>
<text>mRNAs</text>
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<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:42Z</infon>
<location offset="29073" length="18"/>
<text>lipopolysaccharide</text>
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<annotation id="1327">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:51Z</infon>
<location offset="29093" length="3"/>
<text>LPS</text>
</annotation>
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<infon key="score">0.9987564</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29140" length="6"/>
<text>Roquin</text>
</annotation>
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<infon key="score">0.9633093</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29155" length="4"/>
<text>mRNA</text>
</annotation>
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<infon key="score">0.62592363</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:05:58Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="29191" length="8"/>
<text>inactive</text>
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<infon key="score">0.9974112</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:47Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29200" length="5"/>
<text>mRNAs</text>
</annotation>
<annotation id="736">
<infon key="score">0.9992381</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29213" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="737">
<infon key="score">0.9640897</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29231" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="738">
<infon key="score">0.9966534</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29305" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="739">
<infon key="score">0.9888611</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29364" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="740">
<infon key="score">0.9852724</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29376" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="741">
<infon key="score">0.8447384</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:57:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29439" length="10"/>
<text>deficiency</text>
</annotation>
<annotation id="1158">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:54Z</infon>
<location offset="29490" length="11"/>
<text>deletion of</text>
</annotation>
<annotation id="742">
<infon key="score">0.9980139</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29506" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="743">
<infon key="score">0.9953726</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29538" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="744">
<infon key="score">0.9960139</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29595" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="745">
<infon key="score">0.9970066</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="29600" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="746">
<infon key="score">0.99847347</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:56Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29608" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="747">
<infon key="score">0.99569356</infon>
<infon key="type">protein_type</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:55:11Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="29618" length="7"/>
<text>Regnase</text>
</annotation>
<annotation id="1351">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:57:52Z</infon>
<location offset="29813" length="27"/>
<text>double and triple knockouts</text>
</annotation>
<annotation id="748">
<infon key="score">0.99602485</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29895" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="749">
<infon key="score">0.99506897</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="29993" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="750">
<infon key="score">0.992296</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30006" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="751">
<infon key="score">0.5500128</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:57:57Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30061" length="14"/>
<text>reconstitution</text>
</annotation>
<annotation id="752">
<infon key="score">0.99912375</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30081" length="2"/>
<text>WT</text>
</annotation>
<annotation id="753">
<infon key="score">0.9981454</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30084" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="754">
<infon key="score">0.9952649</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30118" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="755">
<infon key="score">0.54457563</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="30185" length="6"/>
<text>active</text>
</annotation>
<annotation id="756">
<infon key="score">0.99797314</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="30192" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="757">
<infon key="score">0.99881476</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30200" length="8"/>
<text>Roquin-1</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>30235</offset>
<text>Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. Both were required for Regnase-1-mediated repression. These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.</text>
<annotation id="758">
<infon key="score">0.9991494</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:36Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30279" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="759">
<infon key="score">0.999046</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:11Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30291" length="8"/>
<text>Roquin-2</text>
</annotation>
<annotation id="760">
<infon key="score">0.8295006</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30333" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="761">
<infon key="score">0.99895585</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="30338" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="762">
<infon key="score">0.99817204</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:44:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30346" length="5"/>
<text>EMSAs</text>
</annotation>
<annotation id="763">
<infon key="score">0.9980009</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30360" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="764">
<infon key="score">0.95859957</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30370" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="765">
<infon key="score">0.99855715</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:20Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30432" length="21"/>
<text>filter binding assays</text>
</annotation>
<annotation id="1256">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="30482" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="766">
<infon key="score">0.99897313</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30550" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1088">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<location offset="30558" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="767">
<infon key="score">0.9221935</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30576" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="768">
<infon key="score">0.997972</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30581" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="769">
<infon key="score">0.99927336</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="30645" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="1218">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="30679" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1290">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<location offset="30682" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="770">
<infon key="score">0.9973216</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:13Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30754" length="23"/>
<text>Regnase-1-binding sites</text>
</annotation>
<annotation id="771">
<infon key="score">0.9932945</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:17Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="30781" length="48"/>
<text>crosslinking and immunoprecipitation experiments</text>
</annotation>
<annotation id="772">
<infon key="score">0.97262865</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30850" length="7"/>
<text>triloop</text>
</annotation>
<annotation id="773">
<infon key="score">0.973413</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:31Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30874" length="34"/>
<text>pyrimidine–purine–pyrimidine loops</text>
</annotation>
<annotation id="774">
<infon key="score">0.99757975</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:28Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30928" length="5"/>
<text>stems</text>
</annotation>
<annotation id="775">
<infon key="score">0.9753175</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:33Z</infon>
<infon key="identifier">SO:</infon>
<location offset="30954" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="776">
<infon key="score">0.7826226</infon>
<infon key="type">gene</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:54:30Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="30980" length="5"/>
<text>Ptgs2</text>
</annotation>
<annotation id="777">
<infon key="score">0.9988354</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31015" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="778">
<infon key="score">0.99926615</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:10:22Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31098" length="9"/>
<text>Regnase-1</text>
</annotation>
<annotation id="779">
<infon key="score">0.72274804</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31123" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="780">
<infon key="score">0.6536611</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:12:40Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31132" length="8"/>
<text>hexaloop</text>
</annotation>
</passage>
<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>31199</offset>
<text>Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.</text>
<annotation id="781">
<infon key="score">0.9875123</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:51:43Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31245" length="12"/>
<text>cis-elements</text>
</annotation>
<annotation id="782">
<infon key="score">0.99735665</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31292" length="3"/>
<text>SLs</text>
</annotation>
<annotation id="783">
<infon key="score">0.99912375</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31302" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="784">
<infon key="score">0.9991041</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31310" length="4"/>
<text>Icos</text>
</annotation>
<annotation id="785">
<infon key="score">0.99302953</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="31430" length="4"/>
<text>mRNA</text>
</annotation>
<annotation id="786">
<infon key="score">0.99902904</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31513" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="787">
<infon key="score">0.9989248</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:52:06Z</infon>
<infon key="identifier">SO:</infon>
<location offset="31520" length="10"/>
<text>loop motif</text>
</annotation>
<annotation id="788">
<infon key="score">0.99890745</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="31580" length="6"/>
<text>Roquin</text>
</annotation>
<annotation id="789">
<infon key="score">0.99247086</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
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<passage>
<infon key="section_type">DISCUSS</infon>
<infon key="type">paragraph</infon>
<offset>31958</offset>
<text>The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.</text>
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<infon key="updated_at">2023-09-15T12:09:56Z</infon>
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<text>3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
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<infon key="updated_at">2023-09-15T12:59:32Z</infon>
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<text>mRNA</text>
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<text>SL</text>
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<infon key="updated_at">2023-09-15T12:21:53Z</infon>
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<location offset="32429" length="3"/>
<text>ROQ</text>
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<annotation id="800">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="32468" length="6"/>
<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32475" length="8"/>
<text>bound to</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
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<location offset="32488" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32493" length="6"/>
<text>3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:13:23Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32536" length="22"/>
<text>secondary binding site</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32563" length="5"/>
<text>dsRNA</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="32572" length="6"/>
<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:17Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32580" length="6"/>
<text>B-site</text>
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<annotation id="808">
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32640" length="5"/>
<text>dsRNA</text>
</annotation>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="32680" length="6"/>
<text>Roquin</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
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<location offset="32698" length="4"/>
<text>RNAs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:59:53Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="32730" length="8"/>
<text>affinity</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:56Z</infon>
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<location offset="32739" length="3"/>
<text>SLs</text>
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<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32809" length="3"/>
<text>RNA</text>
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="32831" length="3"/>
<text>Tnf</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32835" length="3"/>
<text>CDE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
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<location offset="32843" length="3"/>
<text>ADE</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32847" length="2"/>
<text>SL</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32850" length="4"/>
<text>RNAs</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:59:58Z</infon>
<infon key="identifier">DUMMY:</infon>
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<text>crystal lattice</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:18:49Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="32952" length="5"/>
<text>dsRNA</text>
</annotation>
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<infon key="score">0.99879366</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="32965" length="6"/>
<text>B site</text>
</annotation>
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<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="33009" length="6"/>
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<infon key="updated_at">2023-09-15T12:09:56Z</infon>
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<location offset="33059" length="6"/>
<text>3′-UTR</text>
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<infon key="updated_at">2023-09-15T12:17:35Z</infon>
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<location offset="33102" length="7"/>
<text>3′-UTRs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="33163" length="2"/>
<text>SL</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:53Z</infon>
<location offset="33166" length="3"/>
<text>RNA</text>
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<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="33214" length="6"/>
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<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33254" length="6"/>
<text>3′-UTR</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:54:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33268" length="6"/>
<text>Nfkbid</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:32Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="33275" length="4"/>
<text>mRNA</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:07:24Z</infon>
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<location offset="33339" length="13"/>
<text>binding sites</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33417" length="6"/>
<text>Roquin</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:52:01Z</infon>
<location offset="33462" length="12"/>
<text>cis elements</text>
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<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:52:01Z</infon>
<location offset="33539" length="12"/>
<text>cis elements</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:00:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="33563" length="8"/>
<text>affinity</text>
</annotation>
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<infon key="updated_at">2023-09-15T12:06:57Z</infon>
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<location offset="33597" length="6"/>
<text>Roquin</text>
</annotation>
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<infon key="score">0.99905556</infon>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:06:57Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33748" length="6"/>
<text>Roquin</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:19:18Z</infon>
<infon key="identifier">PR:</infon>
<location offset="33758" length="5"/>
<text>MALT1</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33818" length="6"/>
<text>3′-UTR</text>
</annotation>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:13:32Z</infon>
<infon key="identifier">MESH:</infon>
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<text>cross-linking and immunoprecipitation experiments</text>
</annotation>
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<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:13:42Z</infon>
<infon key="identifier">SO:</infon>
<location offset="33974" length="17"/>
<text>RNA-binding sites</text>
</annotation>
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<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:13:35Z</infon>
<infon key="identifier">MESH:</infon>
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<text>structural biology</text>
</annotation>
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<passage>
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<infon key="type">title_1</infon>
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<text>Methods</text>
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<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
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<text>SELEX experiments</text>
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<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>34104</offset>
<text>Selection of Roquin-1-bound RNAs from a random RNA library was performed in three rounds of selection with increased stringency of washing (3 × 100 μl, 4 × 100 μl and 5 × 100 μl washing steps) and with decreased protein concentrations (250, 150 and 50 nM). Before selection, 100 μg recombinant Roquin-1 and Roquin-1 M199R N-terminal protein (residues 2–440) were biotinylated: proteins were incubated for 30 min on ice with 10 × molar excess of EZ-link PEG4-NHS-Biotin (Pierce) in PBS (0.1 mg ml−1). Subsequently, the biotinylated protein was purified via gel filtration (MicroSpin column P6, BioRad) and the loss of protein during the biotinylation procedure was estimated by SDS–PAGE and Coomassie staining. The efficiency of the biotinylation reaction was evaluated after spotting of unlabelled and labelled proteins onto a nitrocellulose membrane. After blocking the membrane with 1% BSA in PBS, it was incubated in streptavidin–PE (R-Phycoerythrin) diluted 1:1,000 in PBS for 30 min at room temperature (RT). Subsequently, the membrane was washed three times with PBS and fluorescence intensity of PE bound to biotinylated protein was determined by fluoroimaging (Raytest, FLA5000, 473 nm, Y510 filter).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>35349</offset>
<text>The RNA startpool containing the 47-nt random sequence as well as the RNA pools for the second and third selection rounds were transcribed in vitro from double-stranded PCR DNA, and protein-bound RNA was isolated and reverse transcribed before PCR amplification, as previously described. Following transcription, the samples were separated on an 8% PAGE, the bands excised and RNA purified. Every round of selection started by combining the RNA pool (400 pmol) with biotinylated protein and incubating the mix for 30 min at 37 °C. Subsequently, binding buffer-equilibrated streptavidin-magnetic beads were added and incubated (10 min, 37 °C) to bind the protein–RNA complexes, followed by washes. By boiling the beads in 0.2 mM EDTA in water for 3 min, protein and RNA molecules were released. After removal of beads, the solution served as template for reverse transcription (One-Step RT-PCR Kit, Qiagen) and from the obtained complementary DNA the RNA pool of the next round of selection was transcribed. The cDNAs from every selection round (startpool, round 1, round 2 and round 3) were used for Index-PCRs to analyse the pool composition at every stage during selection. Comparable amounts of the PCR products were combined to one cDNA library and analysed by Solexa Illumina sequencing.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>36660</offset>
<text>Sequence motif and structural analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>36699</offset>
<text>To identify sequence motifs to which Roquin specifically binds, we counted the number of occurrences of each hexamer (46=4,096 motifs) in the sequences obtained by SELEX. We then generated a data set of randomized sequences of the same nucleotide composition as the SELEX-derived sequences, by permuting the SELEX-derived sequences with a custom script. Finally, we counted the number of occurrences of each hexamer in the set of randomized sequences and computed the log2 ratio of the number of occurrences of each motif in the real and randomized sequence sets. To identify a shared sequence motif in the SELEX patterns that showed the strongest enrichment in our selection experiments, the top 100 patterns were analysed with the Motif-based sequence analysis tool MEME (http://meme-suite.org) using the default settings. This analysis revealed three sequence motifs of which the first is shown in Fig. 1b. For the construction of sequence logos, we screened the obtained nucleotide sequences from SELEX replicate 1 and extracted the nucleotide sequences including the 7-nt flanking regions. Sequence logos were constructed with WebLogo 2.8.2 (http://weblogo.berkeley.edu/).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>37877</offset>
<text>For the Ox40 3′-UTR sequence alignment, we extracted Multiz alignments of 60 Vertebrates from the UCSC mouse GRCm38/mm10 assembly for the genomic region chr4:156,016,498–156,016,520. For each species contained in the alignment, we extracted genomic coordinates of the aligned sequence, extended the coordinates by 10 nt upstream and downstream, and retrieved the extended sequences from the corresponding genome assemblies. Finally, sequences were aligned with ClustalW 2.1 with standard settings and the alignment was visualized using Jalview.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>38428</offset>
<text>To evaluate the structural context the inferred motif is located in, we first appended to the nucleotide sequences obtained from the SELEX experiment the SELEX primers 5′-GGAGAGATGTGAACTT-3′ and 5′-AGTTTCGTGGATGCCAC-3′ to the 5′- and 3′-end, respectively. Next, we screened for sequences that contained the inferred motif and performed secondary structure prediction on those sequences with RNAfold from the ViennaRNA package version 1.8 with parameters '-p -d2'. Next, we used a custom Perl script to parse the base-pairing probability file generated by RNAfold and to calculate an average base-pair probability over all sequences that contained the inferred motif.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>39107</offset>
<text>Production of proteins</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>39130</offset>
<text>Cloning of expression vectors for Roquin-1 ROQ (residues 147–326), ROQ (residues 171–326) and Roquin-1 N-term (residues 2–440) was carried out by standard procedures as described previously. Briefly, PCR-amplified fragments were put into pETM11 and pETTrx1a vectors based on pET24d as provided by the Protein Expression and Purification Facility at Helmholtz Zentrum München. All vectors contained tobacco etch virus (TEV) protease recognition sites for subsequent proteolytic removal of the tags. All length-variable Roquin-1 expression constructs were designed and cloned via restriction sites NcoI (5′) and XhoI (3′). ROQ domain RNA-binding mutants were cloned by Quick change PCR with high-fidelity Phusion DNA polymerase and subsequent treatment with DnpI. Alternatively, we used conventional cloning with a two-step PCR protocol and enzymatic restriction.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>40003</offset>
<text>The Roquin-1 fragments (147–326) and (171–326) were expressed as N-terminal His6-thioredoxin fusion proteins as recently described. Isotope-labelled protein for NMR studies was expressed in M9 minimal medium supplemented with 0.5 g l−1 15N ammonium chloride and 2 g l−1unlabelled or [U-13C] glucose. For the preparation of deuterated proteins, cells were adapted and grown as described previously. Briefly, we used a protocol with stepwise adaptation of cells to deuterium changing buffer from no D2O, low glucose to 50% D2O, low glucose and finally 99.5% D2O with deuterated glucose. The Roquin-1 N-terminal domain (residues 2–440) was expressed and purified essentially as described above for the ROQ domain, but no thioredoxin tag was used. For Roquin-1 N-terminal domain, all expression media and the final buffer contained 100 or 25 μM of zinc chloride, respectively.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>40897</offset>
<text>RNA preparation</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>40913</offset>
<text>RNAs were synthesized and purchased from IBA GmbH (Göttingen, Germany), purified via PAGE followed by two steps of desalting. No major impurities were seen in NMR spectra. Complex formation for crystallography and NMR experiments was achieved by dissolving the lyophilized RNA in water or NMR buffer. This stock solution was snap-cooled by boiling at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. All RNAs were stored at −80 °C, to avoid degradation and thermodynamically favoured duplex formation.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>41461</offset>
<text>Full length and fragments of Ox40 3′-UTR mRNA were produced by in vitro transcription (IVT) from DNA templates harbouring a T7 promoter site either with direct incorporation of α-32P-labelled UTP or subsequent 3′-labelling of purified RNA with γ-32P-labelled ATP. DNA templates were cloned by primer extension PCR. For IVT, 50–150 nM of DNA were incubated with 11 mM magnesium chloride, 8% (w/v) PEG8000, 1.25 mM of each NTP and 0.05 mg ml−1 of T7 polymerase in 1 × standard reaction buffer for 3–5 h at 37 °C. Labelled RNAs were produced in 50 μl reactions and purified via spin columns and directly subjected to EMSA assays. Unlabelled RNAs were produced in reactions of 500–5,000 μl. After IVT, the reactions were separated on 8% denaturing SDS–PAGEs, RNA of interest excised and eluted from the gel using the Elutrap kit (GE Healthcare). After elution, RNAs were dialysed against water and lyophilized. Subsequently, RNAs were dissolved in water and stocks generated by boiling them at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. Labelling for EMSA assays was carried out as for short motifs and described recently. As a modification, dephosphorylation was performed for 30 min and 3′-phosphorylation with γ-32P-labelled ATP and T4 polynucleotide kinase for 90 min for higher efficiency, respectively.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>42857</offset>
<text>NMR spectroscopy</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>42874</offset>
<text>NMR measurements of Roquin-1 ROQ (147–326) and ROQ (171–326) were performed in buffers as described, mixed with 10% D2O. Backbone chemical shift assignments of ROQ (171–326) with 1.1- to 1.2-fold excess of the Ox40 ADE-like SL motif or consensus ADE SL RNAs were recorded with protein concentrations of 350–400 μM. HNCA, HNCACB, HNCO, HNcaCO and 3D 15N-edited NOESY spectra were acquired at 298K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 MHz proton Larmor frequency, equipped with TCI cryogenic probe heads. Spectra of ROQ in complex with Ox40 CDE-like SL RNA and the RNA alone have been reported before. Spectra were processed with Topspin3.2 and analysed with CCPNMR Analysis and Sparky. For RNA motifs, one- and two-dimensional imino NOESY spectra with water-flip-back WATERGATE were recorded at 600–900 MHz, at 278 and 298 K at 150–350 μM RNA concentrations. Sequential assignments were guided by secondary structure predictions with mfold and supported by 15N chemical shifts from natural abundance SOFAST-HMQC experiments.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>43968</offset>
<text>Electrophoretic mobility shift assays</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>44006</offset>
<text>The EMSAs with ROQ domain and individual motifs were performed as described previously. In short, for the binding reaction a mastermix containing transfer RNA, 32P-labelled SL RNA and reaction buffer was prepared and then mixed with dilutions of the recombinant proteins to achieve the indicated protein concentrations. The binding was performed for 10 min at RT or 20 min on ice in 20 μl reaction volume in the presence of 2.5 μg μl−1 tRNA from baker’s yeast (Sigma), 500 pM 32P-labelled RNA, 20 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and 1 μg μl−1 BSA. For the binding reaction of Roquin-1 N-terminal with full-length Ox40 3′-UTRs or fragments thereof, ∼1 pmol of RNA was incubated with protein concentrations between 0 and 1,000 μM in a volume of 20 μl. RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, 0.5 × TBE) at 120 V for 40 min at RT. Gels were then fixed, dried and exposed to a phosphor imager screen.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>45015</offset>
<text>X-ray crystallography</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>45037</offset>
<text>The crystallization experiments for ROQ–RNA complexes were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. The crystals of both, Roquin-1 ROQ (171–326) with Ox40 ADE-like SL motif (22mer, 5′-UCCACACCGUUCUAGGUGCUGG-3′) and with the consensus SELEX-derived ADE SL motif (20mer, 5′-UGACUGCGUUUUAGGAGUUA-3′) were obtained from the same condition: 100 mM Bis-Tris buffer pH 5.5, 200 mM sodium chloride and 25% (v/w) PEG 3350. Crystallization was performed using the sitting-drop vapour-diffusion method at 292 K in 24-well plates and a protein concentration of 12 mg ml−1. The crystals appeared after 1 day. For the X-ray diffraction experiments, the crystals of both co-complexes were mounted in a nylon fibre loop and flash cooled to 100 K in liquid nitrogen. The cryoprotection was performed for 2 s in reservoir solution complemented with 20% (v/v) ethylene glycol. Diffraction data for ROQ Ox40 ADE-like motif was collected on the ID29 beamline (ESRF, Grenoble, France) using a Pilatus 6M at a wavelength of 1.25363 Å. Diffraction data for the ROQ-ADE complex were collected using Pilatus 2M detector at 1.00003 Å wavelength at PXIII beamline at SLS (Villigen, Switzerland). All data sets were indexed and integrated using XDS and scaled using SCALA. Intensities were converted to structure–factor amplitudes using the programme TRUNCATE. Table 1 summarizes data collection and processing statistics for both data sets.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>46524</offset>
<text>Structure determination and refinement</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>46563</offset>
<text>The structure of both ROQ-Ox40 ADE-like SL and ROQ-ADE SL were solved by molecular replacement using the native Roquin-1 ROQ (147–326) structure as a search model (PDB: 4QI0 (ref.)). Model building was performed in COOT. RNA molecules were modelled manually. The refinement of both structures was done in REFMAC5 (ref.) using the maximum-likelihood target function including translation, libration and screw-rotation displacements of a pseudo-rigid body (TLS). For the ROQ-ADE SL structure, non-crystallographic symmetry (NCS) averaging was implemented. The final models are characterized by R and Rfree factors of 21.8 and 25.7% for ROQ-Ox40 ADE-like SL, and 18.6 and 23.4% for ROQ-ADE SL (Table 1), respecively. The stereochemical analysis of both final models was done in PROCHECK and MolProbity. It indicates that there are no residues with generously allowed or unfavourable backbone dihedral angles, and that 99.4% (for ROQ-Ox40 ADE-like SL structure) and 92.3% (for ROQ-ADE SL structure) of all residues are in the core region of the Ramachandran plot.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>47625</offset>
<text>Functional assays</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>47643</offset>
<text>Functional assays determining the Roquin-mediated regulation of Ox40 with different 3′-UTR variants were performed as described previously. In brief, Rc3h1/2−/− mouse embryonic fibroblast (MEF) cells, stably transduced with a doxycycline-inducible Roquin-1-p2A-mCherry construct, were retrovirally infected with Ox40 constructs of different 3′-UTR length or mutation, which led to the expression of Ox40 on the cell surface (CDE-like mutation changing nt 14–16 GCA to CGT, ADE-like mutation changing nt 15–17 from GGT to CCA). Forty-eight hours after infection, the cells were split and one half of the cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 and mCherry, connected via the self-cleaving peptide p2A. Thus, Roquin-expressing cells were marked by mCherry expression. Sixteen to 20 h after induction, the cells were harvested, stained with allophycocyanin (APC)-conjugated anti-Ox40 and analysed by flow cytometry. To compare the Ox40 expression levels achieved by different constructs, the relative Ox40 mean fluorescence intensity (MFI) was determined by dividing the MFI of treated (mCherry+) cells by the MFI of untreated (mCherry−) cells.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>48849</offset>
<text>Mouse experiments</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>48867</offset>
<text>Compound mutant mice with the Rc3h1fl/fl (ref.) and Rc3h2fl/fl (ref.) (denoted Rc3h1/2fl/fl), as well as Cd4-Cre-ERT2 (ref.) and Gt(ROSA)26Sortm1(rtTA*M2)Jae alleles were maintained on a C57BL/6 genetic background. All animals were housed in a pathogen-free barrier facility in accordance with the Ludwig-Maximilians-University München institutional, state and federal guidelines.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>49249</offset>
<text>Generation of overexpression vectors</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>49286</offset>
<text>Expression constructs of Roquin-1 and Ox40 were cloned into a modified pRetroX-Tight vector (Clontech). The puromycine-resistance cassette was removed and a cassette containing attR1-ccdB-attR2 was inserted, to generate a Gateway destination vector. Roquin-1 and Ox40 constructs were inserted by LR reaction (Invitrogen). Any mutants thereof were generated by site-directed mutagenesis.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>49673</offset>
<text>Virus production</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>49690</offset>
<text>Replication-deficient retrovirus production and T-cell transduction was performed as previously described. Briefly, retroviral and packaging plasmids were introduced into HEK293T cells by calcium-phosphate transfection. Forty-eight hours after transfection, cell culture supernatants containing the retrovirus particles were harvested, passed through 0.45-μm filters and stored at −80 °C.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>50085</offset>
<text>Cell isolation and culture</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>50112</offset>
<text>Splenocytes were isolated from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice. CD4+ T cells were isolated by negative selection with magnetic beads according to the manufacturer’s instructions (Stem Cell Technologies). CD4+ T cells were cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 1 × nonessential amino acids (Lonza), 10 mM HEPES pH 7.4 (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen) and 100 U ml−1 penicillin–streptomycin (Invitrogen). Rc3h1/2fl/fl deletion was induced by addition of 4′OH-Tamoxifen (0.3 μM) for 24 h. For TH1 differentiation, CD4+ T cells were cultured in six-well plates pre-coated with goat anti-hamster IgG (MP Biochemicals) and DMEM medium further supplemented with anti-CD3 (0,25 μg ml−1), anti-CD28 (2,5 μg ml−1), IL-12 (10 ng ml−1) and anti-IL-4 (10 μg ml−1) for 40 h. Cells were then infected with retroviral constructs, allowing reconstitution with either Roquin-1, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A, and cultured in IL-2 containing media (20 U ml−1). Forty-eight hours after transduction, the cells were split and one half of cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 WT and Roquin-1 mutants. Twenty-four hours after induction, the cells were harvested for analysis by immunoblot and flow cytometry with the indicated antibodies (1:200 anti-mouse Icos-biotin clone 7E–17G9 (eBioscience); 1:200 Streptavidin-PerCP (Becton Dickinson); 1:200 anti-mouse Ox40-PE clone OX-86 (eBioscience)).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>51678</offset>
<text>Immunoblot analysis</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>51698</offset>
<text>CD4+ T cells were incubated for 15 min on ice with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl2 and protease inhibitor mix without EDTA (Roche) and 1 mM dithiothreitol). Lysate was cleared by centrifugation (10 min, 10 000 g, 4 °C). Immunoblotting was performed by standard protocols with hybridoma supernatants containing monoclonal antibody recognizing Roquin-1 and Roquin-2 (anti-roquin, clone 3F12).</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>52168</offset>
<text>mRNA decay experiments</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>52191</offset>
<text>Hela Tet-Off Advanced Cells (Clontech 631156) were stably transduced with retroviruses expressing different Ox40 constructs. FACS analysis 41 h post transduction revealed similar Ox40 surface expression levels on all five cell samples. After transduction, the cell lines were initially cultured for at least 48 h without doxycycline, to ensure high Ox40-expression levels. For each time point, 400 000 cells were spread on one well in a six-well plate. To switch off Ox40-transcription, doxycycline was supplied with the medium at time point 0. After one washing step with PBS, cells were directly harvested from each well with Trizol before Dox application (0 h), as well as 2, 3 and 4 h after Dox application. RNA was isolated using standard Trizol protocols. Reverse transcription was performed with the Qiagen Quantitect Reverse Transcription Kit following the manufacturer’s protocols. Quantitative PCR was carried out on a Roche Light Cycler 480 using the Light Cycler 480 Probes Master Mix and primer-/probe-combinations from Roches Universal Probe Library. Relative mRNA expression levels were calculated by normalization to the housekeeper gene ywhaz.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_2</infon>
<offset>53364</offset>
<text>Surface plasmon resonance</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>53390</offset>
<text>ROQ–RNA binding experiments were performed on a BIACORE 3000 instrument (Biacore Inc.). ROQ domain was diluted to a final concentration of 35 μg ml−1 in 10 mM HEPES pH 7.0 and chemically immobilized (amine coupling) onto CM5 sensor chips (Biacore Inc.). The RNA samples were diluted in the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.005% Tween 20) to the final concentration of 31.25, 62.5, 125, 250 and 500 nM, and 1 and 2 μM, and injected over the sensor chip surface at 30 μl min−1 at 10 °C. The RNA samples were injected onto the sensor chip from the lowest to the highest concentration. Each RNA-type sample was tested three times with the exception of Mut1–3 two times. Injection of 250 nM RNA was always performed in duplicate within each experiment. To subtract any background noise from each data set, all samples were also run over an unmodified sensor chip surface. Data were analysed using BIAevaluation programme (Biacore Inc.) (Supplementary Fig. 7). For each measurement, the equilibrium dissociation constant was calculated (KD) from steady state binding. The KD from three independent experiments were used to calculate the mean values of these variables and the s.e.m. The results for all tested RNA samples are compared in Table 2.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">title_1</infon>
<offset>54700</offset>
<text>Additional information</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>54723</offset>
<text>Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5F5H and 5F5F for the ROQ-Ox40 ADE-like SL and ROQ-ADE SL, respectively. Chemical shifts of the ROQ-Ox40 ADE-like SL and ROQ-ADE SL have been deposited in the Biological Magnetic Resonance Data Bank under accession codes 26587 and 26588, respectively.</text>
</passage>
<passage>
<infon key="section_type">METHODS</infon>
<infon key="type">paragraph</infon>
<offset>55100</offset>
<text>How to cite this article: Janowski, R. et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat. Commun. 7:11032 doi: 10.1038/ncomms11032 (2016).</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">title_1</infon>
<offset>55278</offset>
<text>Supplementary Material</text>
</passage>
<passage>
<infon key="fpage">160</infon>
<infon key="lpage">165</infon>
<infon key="name_0">surname:Turner;given-names:M.</infon>
<infon key="name_1">surname:Hodson;given-names:D.</infon>
<infon key="pub-id_pmid">22326859</infon>
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<infon key="year">2010</infon>
<offset>58667</offset>
<text>MolProbity: all-atom structure validation for macromolecular crystallography</text>
</passage>
<passage>
<infon key="fpage">e1001674</infon>
<infon key="name_0">surname:Sledzinska;given-names:A.</infon>
<infon key="pub-id_pmid">24115907</infon>
<infon key="section_type">REF</infon>
<infon key="source">PLoS Biol.</infon>
<infon key="type">ref</infon>
<infon key="volume">11</infon>
<infon key="year">2013</infon>
<offset>58744</offset>
<text>TGF-beta signalling is required for CD4(+) T cell homeostasis but dispensable for regulatory T cell function</text>
</passage>
<passage>
<infon key="fpage">725</infon>
<infon key="lpage">733</infon>
<infon key="name_0">surname:Glasmacher;given-names:E.</infon>
<infon key="pub-id_pmid">20639877</infon>
<infon key="section_type">REF</infon>
<infon key="source">Nat. Immunol.</infon>
<infon key="type">ref</infon>
<infon key="volume">11</infon>
<infon key="year">2010</infon>
<offset>58853</offset>
<text>Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>58985</offset>
<text>The authors declare no competing financial interests.</text>
</passage>
<passage>
<infon key="section_type">SUPPL</infon>
<infon key="type">footnote</infon>
<offset>59039</offset>
<text>Author contributions A.S. carried out cloning, protein expression and purification, and NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and SELEX experiments, and functional studies were performed by G.A.H, N.W. and S.B. M.B. and R.B. helped setting up the SELEX experiments and identified patterns from NGS data, on the basis of which A.G. and M.Z. analysed the motifs and secondary structures. T.B. contributed unpublished reagents and advice. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript.</text>
</passage>
<passage>
<infon key="file">ncomms11032-f1.jpg</infon>
<infon key="id">f1</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>59694</offset>
<text>SELEX identifies a novel SL RNA ligand of Roquin-1.</text>
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<text>SL</text>
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<text>RNA</text>
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<text>Roquin-1</text>
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<infon key="type">fig_caption</infon>
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<text>(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. The labels correspond to the versions of the genome assemblies in the UCSC server (see Method section). rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. See also Supplementary Fig. 1.</text>
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<text>Roquin-1</text>
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<text>2–440</text>
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<text>Roquin-1 M199R</text>
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<infon key="updated_at">2023-09-15T12:59:06Z</infon>
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<text>2–440</text>
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<text>ADE</text>
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<text>Ox40</text>
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<infon key="updated_at">2023-09-15T12:09:56Z</infon>
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<text>3′-UTR</text>
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<text>MEME</text>
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<text>SELEX</text>
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<text>RNA</text>
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<location offset="60059" length="4"/>
<text>Ox40</text>
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<infon key="updated_at">2023-09-15T12:17:35Z</infon>
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<location offset="60064" length="7"/>
<text>3′-UTRs</text>
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<annotation id="849">
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<infon key="type">gene</infon>
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<infon key="updated_at">2023-09-15T13:26:08Z</infon>
<infon key="identifier">GENE:</infon>
<location offset="60210" length="3"/>
<text>rn5</text>
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<infon key="type">taxonomy_domain</infon>
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<infon key="updated_at">2023-09-15T13:26:24Z</infon>
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<text>rat</text>
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<infon key="updated_at">2023-09-15T13:26:28Z</infon>
<infon key="identifier">MESH:</infon>
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<text>Rattus novegicus</text>
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<location offset="60321" length="5"/>
<text>SELEX</text>
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<annotation id="853">
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<infon key="updated_at">2023-09-15T12:17:15Z</infon>
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<location offset="60345" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60349" length="3"/>
<text>ADE</text>
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<annotation id="855">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60361" length="4"/>
<text>Ox40</text>
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<annotation id="1134">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="60366" length="3"/>
<text>ADE</text>
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<annotation id="856">
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:25:59Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60375" length="8"/>
<text>hexaloop</text>
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<annotation id="857">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60384" length="2"/>
<text>SL</text>
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<annotation id="858">
<infon key="score">0.99191344</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60437" length="3"/>
<text>ADE</text>
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<annotation id="859">
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60441" length="2"/>
<text>SL</text>
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<annotation id="860">
<infon key="score">0.99405</infon>
<infon key="type">bond_interaction</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:25:29Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="60465" length="24"/>
<text>non-Watson–Crick pairing</text>
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<annotation id="861">
<infon key="score">0.9991272</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
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<text>Ox40</text>
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<location offset="60509" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60520" length="3"/>
<text>Tnf</text>
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<infon key="updated_at">2023-09-15T12:08:49Z</infon>
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<location offset="60524" length="3"/>
<text>CDE</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="60528" length="2"/>
<text>SL</text>
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</passage>
<passage>
<infon key="file">ncomms11032-f2.jpg</infon>
<infon key="id">f2</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>60600</offset>
<text>NMR analysis of the SL RNAs used in this study.</text>
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<infon key="updated_at">2023-09-15T12:11:01Z</infon>
<infon key="identifier">MESH:</infon>
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<text>NMR</text>
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<infon key="updated_at">2023-09-15T12:17:15Z</infon>
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<location offset="60620" length="2"/>
<text>SL</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
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<text>RNAs</text>
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<passage>
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<infon key="type">fig_caption</infon>
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<text>Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). The respective SL RNAs and their base pairs are indicated. Red asterisks indicate NMR signals of the protein. Black asterisks in a indicate a second conformation (see Supplementary Notes). Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. Red nucleotides indicate significant chemical shift changes observed. The dotted green line between G6 and G15 in a highlights a G–G base pair.</text>
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<text>spectra</text>
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<location offset="60714" length="3"/>
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<location offset="60718" length="2"/>
<text>SL</text>
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<text>Ox40</text>
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<text>ADE</text>
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<infon key="annotator">cleaner0</infon>
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<location offset="60744" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T12:07:58Z</infon>
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<location offset="60759" length="4"/>
<text>Ox40</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="60773" length="2"/>
<text>SL</text>
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<infon key="updated_at">2023-09-15T13:29:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60790" length="4"/>
<text>free</text>
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<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="60795" length="4"/>
<text>RNAs</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:29:09Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="60812" length="15"/>
<text>in complex with</text>
</annotation>
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<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="60832" length="8"/>
<text>Roquin-1</text>
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<infon key="updated_at">2023-09-15T12:21:54Z</infon>
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<location offset="60841" length="3"/>
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<location offset="60874" length="2"/>
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<infon key="identifier">MESH:</infon>
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<text>NMR</text>
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<infon key="identifier">MESH:</infon>
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<text>NMR</text>
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<infon key="identifier">DUMMY:</infon>
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<text>signals</text>
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<text>G6</text>
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<infon key="updated_at">2023-09-15T12:41:45Z</infon>
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<text>G15</text>
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<infon key="updated_at">2023-09-15T14:04:18Z</infon>
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<location offset="61335" length="1"/>
<text>G</text>
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<annotation id="885">
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<infon key="type">residue_name</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:04:21Z</infon>
<infon key="identifier">SO:</infon>
<location offset="61337" length="1"/>
<text>G</text>
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<passage>
<infon key="file">ncomms11032-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>61352</offset>
<text>Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.</text>
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<infon key="score">0.99648666</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:00:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61352" length="9"/>
<text>Structure</text>
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<annotation id="887">
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<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="61369" length="8"/>
<text>Roquin-1</text>
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<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="61378" length="3"/>
<text>ROQ</text>
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<annotation id="889">
<infon key="score">0.9990448</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:16:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61389" length="8"/>
<text>bound to</text>
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<infon key="score">0.18962327</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="61398" length="4"/>
<text>Ox40</text>
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<annotation id="1136">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="61403" length="3"/>
<text>ADE</text>
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<annotation id="1260">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<location offset="61412" length="3"/>
<text>RNA</text>
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<passage>
<infon key="file">ncomms11032-f3.jpg</infon>
<infon key="id">f3</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>61417</offset>
<text>(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). Selected RNA bases and protein secondary structure elements are labelled. (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). Only RNA-interacting residues that are different in both structures are shown. Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). The protein chain is shown in turquoise and the RNA is shown in grey. Atoms are colour coded according to charge. (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. Lys259 interacts with the phosphate groups of U10 and U11. (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. In d – f, amino acids are shown in turquoise and blue, nucleotides in grey colour. See also Supplementary Notes and Supplementary Fig. 2.</text>
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<infon key="score">0.9974001</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:20:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61449" length="17"/>
<text>crystal structure</text>
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<annotation id="892">
<infon key="score">0.88668007</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="61474" length="3"/>
<text>ROQ</text>
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<annotation id="893">
<infon key="score">0.99723226</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:30:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61495" length="7"/>
<text>174–325</text>
</annotation>
<annotation id="894">
<infon key="score">0.8609921</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="61518" length="4"/>
<text>Ox40</text>
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<annotation id="1137">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="61523" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1224">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="61532" length="2"/>
<text>SL</text>
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<annotation id="1261">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<location offset="61535" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="895">
<infon key="score">0.94823456</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="61559" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="896">
<infon key="score">0.8175388</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="61649" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1138">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="61654" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1225">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="61663" length="2"/>
<text>SL</text>
</annotation>
<annotation id="897">
<infon key="score">0.99320316</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="61680" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="898">
<infon key="score">0.7903729</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:11Z</infon>
<infon key="identifier">SO:</infon>
<location offset="61684" length="8"/>
<text>hexaloop</text>
</annotation>
<annotation id="899">
<infon key="score">0.99641955</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:14Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61747" length="9"/>
<text>structure</text>
</annotation>
<annotation id="900">
<infon key="score">0.99920934</infon>
<infon key="type">complex_assembly</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:38Z</infon>
<infon key="identifier">GO:</infon>
<location offset="61764" length="11"/>
<text>ROQ-Tnf CDE</text>
</annotation>
<annotation id="901">
<infon key="score">0.9983535</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="61806" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="902">
<infon key="score">0.99855155</infon>
<infon key="type">site</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:50Z</infon>
<infon key="identifier">SO:</infon>
<location offset="61836" length="24"/>
<text>RNA-interacting residues</text>
</annotation>
<annotation id="903">
<infon key="score">0.9811787</infon>
<infon key="type">evidence</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:31:17Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="61888" length="10"/>
<text>structures</text>
</annotation>
<annotation id="904">
<infon key="score">0.802569</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:23:25Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="61958" length="4"/>
<text>RNAs</text>
</annotation>
<annotation id="905">
<infon key="score">0.8209271</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62083" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="906">
<infon key="score">0.99922943</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62110" length="3"/>
<text>U11</text>
</annotation>
<annotation id="907">
<infon key="score">0.9991623</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62118" length="3"/>
<text>U13</text>
</annotation>
<annotation id="908">
<infon key="score">0.88035244</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:58Z</infon>
<infon key="identifier">PR:</infon>
<location offset="62129" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1139">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:20Z</infon>
<location offset="62134" length="3"/>
<text>ADE</text>
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<annotation id="1226">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="62143" length="2"/>
<text>SL</text>
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<annotation id="1262">
<infon key="type">chemical</infon>
<infon key="identifier">CHEBI:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<location offset="62146" length="3"/>
<text>RNA</text>
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<annotation id="909">
<infon key="score">0.99901223</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:24Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62203" length="5"/>
<text>helix</text>
</annotation>
<annotation id="910">
<infon key="score">0.9995128</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:29Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62209" length="2"/>
<text>α4</text>
</annotation>
<annotation id="911">
<infon key="score">0.9995265</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:07:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62213" length="6"/>
<text>Tyr250</text>
</annotation>
<annotation id="912">
<infon key="score">0.9995233</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:32:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62224" length="6"/>
<text>Ser253</text>
</annotation>
<annotation id="913">
<infon key="score">0.9988016</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:32:19Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62259" length="6"/>
<text>strand</text>
</annotation>
<annotation id="914">
<infon key="score">0.99937767</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:36:35Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62266" length="2"/>
<text>β3</text>
</annotation>
<annotation id="915">
<infon key="score">0.9995455</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62270" length="6"/>
<text>Phe255</text>
</annotation>
<annotation id="916">
<infon key="score">0.99955183</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:52Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62281" length="6"/>
<text>Val257</text>
</annotation>
<annotation id="917">
<infon key="score">0.9871703</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="62338" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="918">
<infon key="score">0.5823077</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62450" length="3"/>
<text>ROQ</text>
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<annotation id="919">
<infon key="score">0.99887043</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:40Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62477" length="3"/>
<text>U10</text>
</annotation>
<annotation id="920">
<infon key="score">0.9986286</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62482" length="3"/>
<text>U11</text>
</annotation>
<annotation id="921">
<infon key="score">0.99868125</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62490" length="3"/>
<text>U13</text>
</annotation>
<annotation id="922">
<infon key="score">0.99749124</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:54Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="62501" length="3"/>
<text>RNA</text>
</annotation>
<annotation id="923">
<infon key="score">0.89736986</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:32:32Z</infon>
<infon key="identifier">SO:</infon>
<location offset="62505" length="8"/>
<text>hexaloop</text>
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<annotation id="924">
<infon key="score">0.9991937</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:21Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62515" length="3"/>
<text>U11</text>
</annotation>
<annotation id="925">
<infon key="score">0.9991509</infon>
<infon key="type">residue_name_number</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:39:47Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="62523" length="3"/>
<text>U13</text>
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<text>NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.</text>
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<text>RNA</text>
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<text>(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). Selected shifts of amide resonances are indicated. (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. Gaps indicate prolines. (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). See also Supplementary Notes and Supplementary Fig. 3.</text>
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<text>spectra</text>
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<text>Overlay</text>
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<text>ROQ</text>
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<text>alone</text>
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<text>Ox40</text>
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<text>Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.</text>
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<text>Roquin-1</text>
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<text>Ox40</text>
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<infon key="updated_at">2023-09-15T12:09:56Z</infon>
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<offset>63840</offset>
<text>(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).</text>
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<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="63880" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="974">
<infon key="score">0.9991366</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="63901" length="5"/>
<text>Y250A</text>
</annotation>
<annotation id="975">
<infon key="score">0.9991787</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:05Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="63907" length="6"/>
<text>mutant</text>
</annotation>
<annotation id="976">
<infon key="score">0.99912864</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:21:54Z</infon>
<infon key="identifier">SO:</infon>
<location offset="63914" length="3"/>
<text>ROQ</text>
</annotation>
<annotation id="977">
<infon key="score">0.9992931</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="63944" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1144">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:21Z</infon>
<location offset="63949" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1231">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="63958" length="2"/>
<text>SL</text>
</annotation>
<annotation id="978">
<infon key="score">0.99656075</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:03Z</infon>
<infon key="identifier">PR:</infon>
<location offset="63996" length="3"/>
<text>Tnf</text>
</annotation>
<annotation id="979">
<infon key="score">0.9992507</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64000" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="980">
<infon key="score">0.9995161</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64004" length="2"/>
<text>SL</text>
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<annotation id="981">
<infon key="score">0.99809736</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64176" length="14"/>
<text>Flow cytometry</text>
</annotation>
<annotation id="982">
<infon key="score">0.9991708</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64194" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="983">
<infon key="score">0.99922466</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:50:12Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64203" length="4"/>
<text>Icos</text>
</annotation>
<annotation id="1332">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:04:32Z</infon>
<location offset="64250" length="5"/>
<text>Rc3h1</text>
</annotation>
<annotation id="1336">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:40:24Z</infon>
<location offset="64256" length="3"/>
<text>2fl</text>
</annotation>
<annotation id="1337">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:40:33Z</infon>
<location offset="64260" length="2"/>
<text>fl</text>
</annotation>
<annotation id="1155">
<infon key="type">taxonomy_domain</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:12:08Z</infon>
<location offset="64283" length="4"/>
<text>mice</text>
</annotation>
<annotation id="984">
<infon key="score">0.99808097</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:07Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="64301" length="9"/>
<text>tamoxifen</text>
</annotation>
<annotation id="1333">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:38:23Z</infon>
<location offset="64328" length="5"/>
<text>Rc3h1</text>
</annotation>
<annotation id="1338">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:40:55Z</infon>
<location offset="64334" length="3"/>
<text>2fl</text>
</annotation>
<annotation id="1339">
<infon key="type">gene</infon>
<infon key="identifier">GENE:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:41:04Z</infon>
<location offset="64338" length="2"/>
<text>fl</text>
</annotation>
<annotation id="1340">
<infon key="type">experimental_method</infon>
<infon key="identifier">MESH:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:41:12Z</infon>
<location offset="64341" length="8"/>
<text>deletion</text>
</annotation>
<annotation id="985">
<infon key="score">0.8627631</infon>
<infon key="type">taxonomy_domain</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:04:28Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64451" length="10"/>
<text>retrovirus</text>
</annotation>
<annotation id="986">
<infon key="score">0.9809732</infon>
<infon key="type">chemical</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:52:23Z</infon>
<infon key="identifier">CHEBI:</infon>
<location offset="64475" length="11"/>
<text>doxycycline</text>
</annotation>
<annotation id="987">
<infon key="score">0.99905056</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64518" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="988">
<infon key="score">0.999236</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:00Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64527" length="2"/>
<text>WT</text>
</annotation>
<annotation id="989">
<infon key="score">0.99354047</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64531" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="990">
<infon key="score">0.99891853</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:16Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64540" length="5"/>
<text>Y250A</text>
</annotation>
<annotation id="991">
<infon key="score">0.9958498</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64549" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="992">
<infon key="score">0.9989888</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:27Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64558" length="5"/>
<text>K220A</text>
</annotation>
<annotation id="993">
<infon key="score">0.99904567</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:47:32Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64565" length="5"/>
<text>K239A</text>
</annotation>
<annotation id="994">
<infon key="score">0.9991043</infon>
<infon key="type">mutant</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:08:08Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64575" length="5"/>
<text>R260A</text>
</annotation>
<annotation id="995">
<infon key="score">0.9720185</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:07Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64581" length="7"/>
<text>mutants</text>
</annotation>
</passage>
<passage>
<infon key="file">ncomms11032-f6.jpg</infon>
<infon key="id">f6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_title_caption</infon>
<offset>64624</offset>
<text>Functional importance of Roquin-1 target motifs in cells.</text>
<annotation id="996">
<infon key="score">0.99915963</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64649" length="8"/>
<text>Roquin-1</text>
</annotation>
</passage>
<passage>
<infon key="file">ncomms11032-f6.jpg</infon>
<infon key="id">f6</infon>
<infon key="section_type">FIG</infon>
<infon key="type">fig_caption</infon>
<offset>64682</offset>
<text>(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). Arrows indicate the individual binding events to either motif. It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). Error bars represent the mean of technical duplicates in one experiment. mRNA half-life times were calculated with Graph Pad Prism. Data are representative of two experiments with similar results.</text>
<annotation id="997">
<infon key="score">0.99841917</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64702" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="998">
<infon key="score">0.9961896</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64707" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="1341">
<infon key="type">protein_state</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:41:44Z</infon>
<location offset="64718" length="9"/>
<text>truncated</text>
</annotation>
<annotation id="999">
<infon key="score">0.8903231</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:11Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64728" length="7"/>
<text>mutated</text>
</annotation>
<annotation id="1000">
<infon key="score">0.99823916</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:13Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64765" length="4"/>
<text>EMSA</text>
</annotation>
<annotation id="1001">
<infon key="score">0.99877816</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64816" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1002">
<infon key="score">0.9985067</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:58:18Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64837" length="4"/>
<text>EMSA</text>
</annotation>
<annotation id="1003">
<infon key="score">0.99905396</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64890" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="1004">
<infon key="score">0.9968333</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:59:06Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64927" length="5"/>
<text>2–440</text>
</annotation>
<annotation id="1005">
<infon key="score">0.9991339</infon>
<infon key="type">protein_state</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:56:48Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="64958" length="9"/>
<text>wild-type</text>
</annotation>
<annotation id="1006">
<infon key="score">0.9980616</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="64968" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1007">
<infon key="score">0.99715513</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="64973" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="1008">
<infon key="score">0.7102474</infon>
<infon key="type">experimental_method</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:06:33Z</infon>
<infon key="identifier">MESH:</infon>
<location offset="64997" length="9"/>
<text>mutations</text>
</annotation>
<annotation id="1093">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:08:49Z</infon>
<location offset="65014" length="3"/>
<text>CDE</text>
</annotation>
<annotation id="1232">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="65023" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1145">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:21Z</infon>
<location offset="65031" length="3"/>
<text>ADE</text>
</annotation>
<annotation id="1233">
<infon key="type">structure_element</infon>
<infon key="identifier">SO:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:17:15Z</infon>
<location offset="65040" length="2"/>
<text>SL</text>
</annotation>
<annotation id="1009">
<infon key="score">0.9990656</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:58:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65051" length="3"/>
<text>SLs</text>
</annotation>
<annotation id="1010">
<infon key="score">0.99915844</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:37Z</infon>
<infon key="identifier">PR:</infon>
<location offset="65275" length="8"/>
<text>Roquin-1</text>
</annotation>
<annotation id="1011">
<infon key="score">0.9939894</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:09:56Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65293" length="6"/>
<text>3′-UTR</text>
</annotation>
<annotation id="1012">
<infon key="score">0.997663</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="65336" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1353">
<infon key="type">evidence</infon>
<infon key="identifier">DUMMY:</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T14:02:02Z</infon>
<location offset="65341" length="35"/>
<text>MFI normalized to expression levels</text>
</annotation>
<annotation id="1013">
<infon key="score">0.9985461</infon>
<infon key="type">protein</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T12:07:59Z</infon>
<infon key="identifier">PR:</infon>
<location offset="65386" length="4"/>
<text>Ox40</text>
</annotation>
<annotation id="1014">
<infon key="score">0.9986028</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:52:10Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65391" length="3"/>
<text>CDS</text>
</annotation>
<annotation id="1015">
<infon key="score">0.99770576</infon>
<infon key="type">structure_element</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:52:14Z</infon>
<infon key="identifier">SO:</infon>
<location offset="65437" length="3"/>
<text>CDS</text>
</annotation>
<annotation id="1016">
<infon key="score">0.99085015</infon>
<infon key="type">residue_range</infon>
<infon key="annotator">cleaner0</infon>
<infon key="updated_at">2023-09-15T13:42:02Z</infon>
<infon key="identifier">DUMMY:</infon>
<location offset="65442" length="4"/>
<text>1–40</text>
</annotation>
<annotation id="1017">
<infon key="score">0.9923784</infon>
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<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><bold>ROQ-</bold><italic><bold>Ox40</bold></italic>
<bold>ADE-like SL</bold></th><th align="center" valign="top" charoff="50"><bold>ROQ-ADE SL</bold></th></tr></thead><tbody valign="top"><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Data collection</italic></td></tr><tr><td align="left" valign="top" charoff="50"> space group</td><td align="center" valign="top" charoff="50"><italic>P</italic>2<sub>1</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50"><italic>P</italic>2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="center" valign="top" charoff="50">89.66, 115.79, 42.61</td><td align="center" valign="top" charoff="50">72.90, 89.30, 144.70</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>α, β, γ</italic> (°)</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">50–2.23 (2.29–2.23)</td><td align="center" valign="top" charoff="50">50–3.0 (3.08–3.00)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>merge</sub></td><td align="center" valign="top" charoff="50">5.9 (68.3)</td><td align="center" valign="top" charoff="50">14.8 (93.8)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/σ<italic>I</italic></td><td align="center" valign="top" charoff="50">14.9 (2.1)</td><td align="center" valign="top" charoff="50">16.7 (3.1)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="center" valign="top" charoff="50">98.7 (97.7)</td><td align="center" valign="top" charoff="50">99.9 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy</td><td align="center" valign="top" charoff="50">3.9 (3.7)</td><td align="center" valign="top" charoff="50">13.2 (12.7)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">2.23</td><td align="center" valign="top" charoff="50">3.00</td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections</td><td align="center" valign="top" charoff="50">21,018</td><td align="center" valign="top" charoff="50">18,598</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">21.8/25.7</td><td align="center" valign="top" charoff="50">18.6/23.4</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> No. atoms</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">2,404</td><td align="center" valign="top" charoff="50">4,820</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">894</td><td align="center" valign="top" charoff="50">1,708</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">99</td><td align="center" valign="top" charoff="50">49</td></tr><tr><td align="left" valign="top" charoff="50"><italic> B</italic>-factor overall</td><td align="center" valign="top" charoff="50">47.2</td><td align="center" valign="top" charoff="50">60.4</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Root mean squared deviations</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.006</td><td align="center" valign="top" charoff="50">0.014</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="center" valign="top" charoff="50">1.07</td><td align="center" valign="top" charoff="50">1.77</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50">Ramachandran plot</td></tr><tr><td align="left" valign="top" charoff="50"> Most favoured (%)</td><td align="center" valign="top" charoff="50">98.6</td><td align="center" valign="top" charoff="50">99.8</td></tr><tr><td align="left" valign="top" charoff="50"> Additional allowed (%)</td><td align="center" valign="top" charoff="50">1.4</td><td align="center" valign="top" charoff="50">0.2</td></tr></tbody></table>
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<text> ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection space group P21212 P212121 Cell dimensions a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00) Rmerge 5.9 (68.3) 14.8 (93.8) I/σI 14.9 (2.1) 16.7 (3.1) Completeness (%) 98.7 (97.7) 99.9 (99.9) Redundancy 3.9 (3.7) 13.2 (12.7) Refinement Resolution (Å) 2.23 3.00 No. reflections 21,018 18,598 Rwork/Rfree 21.8/25.7 18.6/23.4 No. atoms Protein 2,404 4,820 Ligand/ion 894 1,708 Water 99 49 B-factor overall 47.2 60.4 Root mean squared deviations Bond lengths (Å) 0.006 0.014 Bond angles (°) 1.07 1.77 Ramachandran plot Most favoured (%) 98.6 99.8 Additional allowed (%) 1.4 0.2 </text>
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<collection><source>PMC</source><date>20201216</date><key>pmc.key</key><document><id>4806292</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep23641</infon><infon key="article-id_pii">srep23641</infon><infon key="article-id_pmc">4806292</infon><infon key="article-id_pmid">27009356</infon><infon key="elocation-id">23641</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Klima;given-names:Martin</infon><infon key="name_1">surname:Tóth;given-names:Dániel J.</infon><infon key="name_10">surname:Humpolickova;given-names:Jana</infon><infon key="name_11">surname:Nencka;given-names:Radim</infon><infon key="name_12">surname:Veverka;given-names:Vaclav</infon><infon key="name_13">surname:Balla;given-names:Tamas</infon><infon key="name_14">surname:Boura;given-names:Evzen</infon><infon key="name_2">surname:Hexnerova;given-names:Rozalie</infon><infon key="name_3">surname:Baumlova;given-names:Adriana</infon><infon key="name_4">surname:Chalupska;given-names:Dominika</infon><infon key="name_5">surname:Tykvart;given-names:Jan</infon><infon key="name_6">surname:Rezabkova;given-names:Lenka</infon><infon key="name_7">surname:Sengupta;given-names:Nivedita</infon><infon key="name_8">surname:Man;given-names:Petr</infon><infon key="name_9">surname:Dubankova;given-names:Anna</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>121</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1035</offset><text>Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. These structures are essential for effective viral replication. Recently, highly specific PI4KB inhibitors were developed as potential antivirals.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2112</offset><text>PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2697</offset><text>Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3754</offset><text>We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4156</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4164</offset><text>ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4240</offset><text>In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5275</offset><text>Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6446</offset><text>Structural analysis of the ACBD3:PI4KB complex</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6493</offset><text>Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6926</offset><text>For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). Assignments obtained for non-exchangeable side-chain signals were over 99% complete. The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. Structural statistics for the final water-refined sets of structures are shown in SI Table 1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8698</offset><text>This structure revealed that the Q domain forms a two helix hairpin. The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9580</offset><text>To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10144</offset><text>ACBD3 efficiently recruits the PI4KB enzyme to membranes</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10201</offset><text>We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10759</offset><text>We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12033</offset><text>We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13272</offset><text>ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13368</offset><text>To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. We therefore designed a more physiologically relevant experiment. For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. This enabled visualization of the kinase reaction using a confocal microscope. We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). We conclude that enzyme activation proceeds through a membrane recruitment mechanism.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>14664</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>14675</offset><text>Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>15258</offset><text>The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>16782</offset><text>Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. We show that these proteins interact directly with a Kd value in the submicromolar range. The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>18118</offset><text>+RNA viruses replicate at specific PI4P-enriched membranous compartments. These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>19131</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19153</offset><text>Plasmid construction, protein expression, and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19212</offset><text>All proteins used in this study were recombinant and were expressed in E. coli using previously developed protocols. Briefly, full-length human ACBD3 (UniProtKB entry Q9H3P7) and PI4KB (UniProtKB entry Q9UBF8, isoform 1) lipid kinase and their deletion mutants were cloned into a previously modified pRSFD vector (Novagen) that already contained an N-terminal 6xHis tag followed by a GB1 solubility tag and TEV protease cleavage site. Mutations were generated using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). The plasmids used are listed in the SI (SI Table 2). The proteins were expressed in E. coli BL21 Star cells as previously described. Upon overnight expression in autoinduction media bacterial cells were harvested and lysed in lysis buffer (50 mM Tris pH 8, 300 mM NaCl, 3 mM β-mercaptoethanol, 20 mM imidazole, 10% glycerol). The lysate was incubated with the Ni-NTA resin (Macherey-Nagel) and then extensively washed with the lysis buffer. The protein was eluted with the lysis buffer supplemented with 300 mM imidazole. When appropriate, tags were removed with TEV protease, and the protein was further purified using the size exclusion chromatography on Superdex 75 or Superdex 200 columns (GE Healthcare) in SEC buffer (10 mM Tris pH 8, 200 mM NaCl, 3 mM β-ME). Proteins were concentrated to 1–5 mg/ml (measured spectroscopically) and stored at −80 °C until needed.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20638</offset><text>In vitro pull-downs</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20658</offset><text>Ni-NTA sepharose beads (Macherey-Nagel) were mixed with both binding partners (one of which was tagged with an N-terminal 6xHis tag) at a final concentration of 1 μM in a final volume of 200 μL binding buffer (30 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, and 1 mM TCEP). After 30 min incubation at 4 °C the beads were washed twice with 200 μL of the binding buffer, and total protein was directly eluted with the Laemmli sample buffer and analyzed by SDS-PAGE.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21143</offset><text>SPR (Surface plasmon resonance) and AUC (Analytical ultracentrifugation)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21216</offset><text>PI4KB was chip-immobilized as detailed in the SI. Afterwards, the ACBD3 protein was injected in a series of concentrations for 3 min and then dissociation was monitored for another 5 min. The data were fit to a single-exponential model. Rate constants of association and dissociation were obtained by fitting the observed change in resonance signal using the following equations:</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21600</offset><text>where c is the protein concentration, t is time, kon is the association rate constant, koff is the dissociation rate constant, D1 and D2 are the linear drift terms, and Ras, Rdis, R0, R1, and Rmax are corresponding changes in the relative response signal.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21856</offset><text>AUC was used to perform sedimentation velocity experiments using a ProteomeLab XL-I Beckman Coulter analytical ultracentrifuge equipped with an AN50Ti rotor. All measurements were performed in 10 mM Tris pH 8, 200 mM NaCl, and 3 mM β-mercaptoethanol at 20 °C and 48000 rpm. All data were collected using an absorbance (230 nm and 280 nm) optical system. Data analysis was performed with the SEDFIT package and data were analyzed using a sedimentation coefficient distribution model c(s).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22360</offset><text>In vitro kinase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22382</offset><text>In vitro kinase activity was measured using a bioluminescent ADP-Glo assay (Promega) as described previously. Briefly, reactions were carried out in a total volume of 5 μL in 384-well plates by diluting the indicated amounts of the PI4KB enzyme and/or ACBD3 protein into the kinase buffer (20 mM Tris pH 7.5, 5 mM MgCl2, 0.2% Triton-X100, 0.1 mg/mL BSA, 2 mM DTT, 50 μM phosphatidylinositol). Reaction was initiated by adding ATP to a final concentration of 100 μM. Samples were incubated for 60 min at 25 °C and the amount of hydrolyzed ATP was measured according to the manufacturer’s protocol using a TECAN infinite M 1000 plate reader.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23046</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23063</offset><text>NMR spectra were acquired at 25 °C on a 600 MHz and 850 MHz Bruker Avance spectrometers, both of which were equipped with a triple-resonance (15N/13C/1H) cryoprobe. The sample volume was 0.35 mL, with a 280 μM concentration for the free Q domain and a 470 μM concentration for the ACBD3:PI4KB complex in the NMR buffer (25 mM sodium phosphate pH 6.5, 100 mM NaCl, 1 mM TCEP, 0.01% NaN3), 5% D2O/95% H2O. A series of double- and triple-resonance spectra were recorded to determine essentially complete sequence-specific resonance backbone and side-chain assignments. Constraints for 1H-1H distance required to calculate the structure of free Q domain and ACBD3:PI4KB complex were derived from 3D 15N/1H NOESY-HSQC and 13C/1H NOESY-HMQC, which were acquired using a NOE mixing time of 100 ms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23877</offset><text>The families of converged structures for the ACBD3:PI4KB complex and free Q domain were calculated using standard software as detailed in the SI. The structures with the lowest total energy were selected and validated. The statistics for the resulting structures are summarized in SI Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24170</offset><text>Protein labeling with fluorescent dyes</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24209</offset><text>PI4KB was labeled on native cysteine residues. Briefly, pure recombinant protein was incubated overnight at 4 °C with a 3x molar excess of Alexa 488 C5 maleimide (Life Technologies). The reaction was quenched by adding 10 mM β-mercaptoethanol (βME) and the protein was repurified by size exclusion chromatography.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24530</offset><text>Giant Unilamellar Vesicle Preparation and Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24580</offset><text>Giant Unilamellar Vesicles (GUVs) composed of POPC (54.9 mol %), POPS (10 mol %), cholesterol (20 mol %), PI (10 mol %), DGS-NTA(Ni) [1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) ] (5 mol %) (Avanti Polar lipids), and ATTO647N-DOPE (0.1 mol %) (ATTO-TEC GmbH) were prepared by electroformation as described previously, please see SI.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24979</offset><text>Live Cell Imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24997</offset><text>COS-7 cells were plated onto 29-mm-diameter poly-L-Lysine coated glass bottom dishes (In Vitro Scientific) at 100,000 cells/well density and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. The plasmids are described in SI Table 2. 24 hr post transfection, COS-7 cells were washed with a modified Krebs-Ringer solution (10 mM Na-HEPES pH 7.4, 120 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose) in the same dish and were imaged using an LSM 710 confocal microscope (Carl Zeiss MicroImaging) with a 63 × 1.4-numerical-aperture planapochromatic objective. For ceramide uptake experiments, COS-7 cells were loaded with 0.05 μM BODIPY® FL C5-Ceramide (Molecular Probes, ThermoFisher Scientific) complexed with BSA in modified Krebs-Ringer solution at room temperature for 20 min. Cells were then washed three times and imaged using the above mentioned settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25988</offset><text>Immunofluorescent imaging</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26014</offset><text>COS-7 cells were plated onto 25-mm-diameter poly-L-Lysine coated circular glass coverslips in six-well plates (100,000 cells/well), and transfected using the Lipofectamine2000 reagent (Invitrogen) with plasmid DNAs (0.5–1 mg/well) according to manufacturer’s instructions. Twenty four hours post transfection, cells were washed with PBS, fixed with 4% paraformaldehyde, stained with mouse anti-PI4KB primary antibody (BD Transduction Laboratories, 1:500 dilution) and then after washing with PBS stained with Alexa Fluor 647 conjugated donkey anti-mouse secondary antibody (Molecular Probes, ThermoFisher Scientific, 1:500 dilution). Cover slips were mounted and observed with the above mentioned microscopy settings.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>26738</offset><text>HD exchange</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26750</offset><text>Hydrogen/deuterium exchange was performed as previously described with the following modifications. The exchange was done in 10 mM Tris-HCl pD 8.0, 200 mM NaCl at 20 °C. Protein concentration during the exchange was 1 μM. Aliquots (50 μL) were removed after 10, 20, 60, 120, 600, 1800, and 3600 s and the exchange was quenched by the addition of 50 μL of 0.25 M glycine-HCl pH 2.3 and rapid freezing in liquid nitrogen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27188</offset><text>Prior to the analysis each sample was quickly thawed and injected onto an immobilized rhizopuspepsin column (bed volume 66 μL). Digestion was driven by a flow of 0.4% formic acid in water at a flow rate of 100 μL/min (LC-20AD pump, Shimadzu). The resulting peptides were trapped and desalted online on a peptide microtrap (Optimize Technologies). After a desalting step (3 min), peptides were separated using a linear gradient of 10–25% buffer B for 2 min, followed by a quick jump to 99% buffer B (buffer A = 0.4% formic acid/2% acetonitrile in water; buffer B = 95% acetonitrile/0.4% formic acid in water). The outlet of the LC system was interfaced to an electrospray ionization source of a Fourier transform ion cyclotron resonance mass spectrometer (12 T SolariX XR, Bruker Daltonics). Exchange was followed on 32 peptides from PI4KB (N) and 26 peptides from ACBD3(Q), covering in both cases 100% of the protein sequence. Peptides were identified by LC-MS/MS and MASCOT search against a database containing the sequences of the studied proteins. Data from H/D exchange were analyzed by program DeutEx written in the laboratory (unpublished).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>28355</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28378</offset><text>Accession codes: The structures and assigned chemical shifts for the free Q domain and the ACBD3:PI4KB complex were deposited in PDB database under accession codes 2N72 and 2N73, and BMRB database under accession codes 25790 and 25791.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28614</offset><text>How to cite this article: Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Sci. 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ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. (B) In vitro pull-down assay. Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. The asterisks mark the bands corresponding to specific interactions. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. SPR analysis of the PI4KB binding to immobilized ACBD3. Sensorgrams for four concentrations of PI4KB are shown.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35540</offset><text>Structural analysis of the ACBD3:PI4KB complex.</text></passage><passage><infon key="file">srep23641-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35588</offset><text>(A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. ACBD3 is shown in magenta and PI4KB in orange. (C) Top view of the kinase helix. The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. Inputs and bound proteins were analyzed on SDS gels and stained with Coomassie Blue. Cropped gels ran the same experimental conditions are shown. Please, see SI Fig. 9 for original full-length gels.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>36896</offset><text>ACBD3 is sufficient to recruit the PI4KB kinase to membranes.</text></passage><passage><infon key="file">srep23641-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>36958</offset><text>(A) GUVs recruitment assay. Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. (B) Golgi displacement experiment. Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. Middle panel: The same experiment performed with GFP alone. Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. Middle panel – The same experiment performed with mRFP-FKBP alone. Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. Right – The same experiment performed using the H264A Q domain mutant.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>38600</offset><text>ACBD3 indirectly increases the activity of PI4KB.</text></passage><passage><infon key="file">srep23641-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>38650</offset><text>(A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. The error bars stand for SEM based on three independent experiments (also SI Fig. 6).</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>39617</offset><text>Pseudoatomic model of the PI4KB multiprotein complex assembly.</text></passage><passage><infon key="file">srep23641-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39680</offset><text>PI4KB in orange, Rab11 in purple, ACBD3 in blue. The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation.</text></passage></document></collection>
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<collection><source>PMC</source><date>20201215</date><key>pmc.key</key><document><id>4817029</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep23473</infon><infon key="article-id_pii">srep23473</infon><infon key="article-id_pmc">4817029</infon><infon key="article-id_pmid">27032335</infon><infon key="elocation-id">23473</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Liberato;given-names:Marcelo V.</infon><infon key="name_1">surname:Silveira;given-names:Rodrigo L.</infon><infon key="name_10">surname:Polikarpov;given-names:Igor</infon><infon key="name_2">surname:Prates;given-names:Érica T.</infon><infon key="name_3">surname:de Araujo;given-names:Evandro A.</infon><infon key="name_4">surname:Pellegrini;given-names:Vanessa O. A.</infon><infon key="name_5">surname:Camilo;given-names:Cesar M.</infon><infon key="name_6">surname:Kadowaki;given-names:Marco A.</infon><infon key="name_7">surname:Neto;given-names:Mario de O.</infon><infon key="name_8">surname:Popov;given-names:Alexander</infon><infon key="name_9">surname:Skaf;given-names:Munir S.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>105</offset><text>Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1053</offset><text>The production of biofuels from renewable sources is an important element of the global strategy for generating sustainable energy with reduced environmental impact. Current technologies for obtaining liquid biofuels and green chemicals rely on the enzymatic digestion of lignocellulosic biomass from a variety of feedstocks. Plant biomass-the most abundant source of carbohydrates on Earth-is primarily composed of cellulose microfibrils surrounded by a hydrated heteropolymeric matrix of hemicellulose and lignin. Plant biomass may be subjected to thermo-chemical pretreatments and enzymatic reactions to produce soluble fermentable sugars.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1696</offset><text>The canonical model of hydrolytic degradation of cellulose requires at least three classes of enzymes. Cellobiohydrolases (CBHs) processively cleave the glycosidic bonds at the reducing and non-reducing ends of cellulose chains in crystalline regions to produce cellobiose. Endoglucanases (EGs) introduce random cuts in the amorphous regions of cellulose and create new chain extremities for CBH attack; thus, these enzymes act synergistically. The released cellobiose molecules are then enzymatically converted into glucose by β-glucosidases.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2243</offset><text>The molecular architecture of glycoside hydrolases (GHs) frequently consists of a catalytic domain (CD), where hydrolysis occurs, and one or more ancillary modules (AMs), which are usually connected by less structured linkers. The most common type of AMs are carbohydrate-binding modules (CBMs), which are able to recognize and bind specific carbohydrate chains. Generally distinct and independent structural domains, the CBMs facilitate carbohydrate hydrolysis by increasing the local concentration of enzymes at the surface of insoluble substrates, thereby targeting the CD component to its cognate ligands. CBMs might also disrupt the crystalline structure of cellulose microfibrils, although the underlying mechanism remains poorly understood. Thus, CBMs enhance the accessibility of CDs to carbohydrate chains to improve enzymatic activity, making them important candidates for the development of effective biomass-degrading enzymes in industrial settings.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3205</offset><text>Although there are examples of active GHs that lack AMs, the majority of the enzymes depend on AMs for activity. In several cases, CBMs were shown to extend and complement the CD substrate-binding site in multimodular carbohydrate-active enzymes, such as endo/exocellulase E4 from Thermobifida fusca, chitinase B from Serratia marcescens, a starch phosphatase from Arabidopsis thaliana and a GH5 subfamily 4 (GH5_4) endoglucanase from Bacillus halodurans (BhCel5B). A pioneer work of Sakon et al. revealed that rigid structural extension of the GH9 CD by a type C CBM3 imprints a processive mode of action to this endoglucanase. Further publications showed that CBM-based structural extensions of the active site are important for substrate engagement and recognition.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3974</offset><text>Recently, Venditto et al. reported the X-ray structure of the tri-modular GH5_4 endoglucanase from Bacillus halodurans (31% sequence identity to BlCel5B), with the CBM46 extension of the active site appended to the CD via an immunoglobulin (Ig)-like module. Removal of the CBM46 caused a ~60-fold reduction of the activity of the enzyme against β-glucans, but showed little or no effect against xyloglucan hydrolysis. Moreover, the CBM46 mediated a significant increase in the BhCel5B activity in plant cell wall settings. Modeling of cellotriose in the negative subsites of the active site of BhCel5B demonstrated the structural conservation of the -1 position, but provided little information about direct interactions between CBM46 and the substrate. It was speculated that β-1,3 kink of the β-glucan might allow the ligand to reach for the CBM46, whereas pure β-1,4 linkages in the backbone of xyloglucan chains would restrict binding to the CD, thus explaining the lack of influence of the CBM46 on the enzymatic activity of BhCel5B against xyloglucans in solution. It was also argued that the CBM46 could potentialize the activity by driving BhCel5B towards xyloglucan-rich regions in the context of the plant cell walls, but no large-scale conformational adjustments of the AMs have been shown to occur or suggested to take part in the enzymatic activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5349</offset><text>The mechanisms of ligand binding mediated by large-scale conformational changes in proteins following the induced-fit or conformational selection models have recently attracted considerable attention. Although initially introduced as contradictory theories, these two limiting cases can be unified considering the flux description concept or the extended conformational selection model. While local ligand-induced conformational adjustments have been reported for carbohydrate-active enzymes, cognate ligands recognition and hydrolysis mediated by a large-scale conformational mobility of distinct domains in multidomain settings is uncommon for endoglucanases.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6011</offset><text>Here, we report the crystal structure of a full-length GH5_4 enzyme from Bacillus licheniformis (BlCel5B) that exhibits two AMs (Ig-like module and CBM46) appended to the CD. We structurally and functionally characterize the enzyme using a combination of protein crystallography, small-angle X-ray scattering (SAXS), molecular dynamics computer simulations and site-directed mutagenesis, and show that the AMs and their conformational mobility are essential for the enzymatic activity of BlCel5B. We find that the large-scale conformational adjustments of the distal CBM46 mediated by the Ig-like hinge domain are crucial in active-site assembly for optimal substrate binding and hydrolysis. We propose that the BlCel5B conformational selection/induced-fit mechanism of hydrolysis represents a novel paradigm that applies to several GH5_4 members and, possibly, to a number of other multidomain GHs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6911</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6919</offset><text>BlCel5B Crystal Structure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6945</offset><text>BlCel5B crystals in the substrate-free form and complexed with cellopentaose (C5) were obtained and diffracted to 1.7 Å and 1.75 Å resolutions, respectively (Supplementary Table 1). The substrate-free and complexed structures exhibited no substantial conformational differences (with the exception of the substrate). Because of minor variations in the loops located distal to the substrate-binding site, a root mean squared deviation (rmsd) of 0.33 Å between the complexed and substrate-free structures was observed. A single protein chain occupies the asymmetric unit, and most of the residues were built, with the exception of the first 17 residues and those in the loop between L398 and P405 due to weak electron density.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7679</offset><text>The BlCel5B structure comprises three distinct domains: an N-terminal CD (residues 18 to 330), an Ig-like module (residues 335 to 428) and a family 46 CBM (residues 432 to 533) (Fig. 1A,B). Similarly to other members of the GH5 family, the CD of BlCel5B has a typical TIM barrel fold with eight inner β-strands and eight outer α helices that are interconnected by loops and three short α helices. Very short linkers, D429-D430-P431 and V331-P332-N333-A334, connect the CBM46 to the Ig-like module and the Ig-like module to the CD, respectively. Both Ig-like module and CBM46 have a β-sandwich fold composed of two β-sheets of four and three antiparallel β-strands interconnected by loops and a short α helix between strands β3 and β4 (Fig. 1C). A structural comparison between the Ig-like module and the CBM46 using the Dali server yielded an rmsd of 2.3 Å and a Z-score of 10.2. However, despite their structural resemblance, these modules share only 17% sequence identity. A structure-based search performed using the same server showed that the Ig-like module is similar to the Ig-like module from a recently solved crystal structure of a tri-modular GH5_4 enzyme from Bacillus halodurans, BhCel5B, with rmsd = 1.3 Å and Z-score = 15.3. The CBM46 from BhCel5B is the most structurally similar to BlCel5B CBM46, with rmsd = 1.6 Å and Z-score = 12.4. The sequence identity relative to BhCel5B, however, is low (28% for Ig-like and 25% for CBM46).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9161</offset><text>The Ig-like module, adjacent to the CD, contains only one tyrosine (Y367) exposed to solvent and no tryptophan residues. Because aromatic residues play a major role in glucose recognition, this observation suggests that substrate binding may not be the primary function of Ig-like module. In contrast, the CBM46 has three tryptophan residues, two of which face the CD substrate binding site (Fig. 1A), indicating that it may be actively engaged in the carbohydrate binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9635</offset><text>Electron density maps clearly reveal the presence of a cellotetraose (C4) and not a soaked cellopentaose (C5) in the CD negative substrate-binding subsites (Fig. 1D), indicating that BlCel5B is catalytically active in the crystal state and able to cleave a C5 molecule. The lack of electron density verifies the absence of the fifth glucose moiety from the soaked C5, and a closer inspection of the structure confirmed that the presence of a fifth glucose unit would be sterically hindered by the catalytic residues on the reducing end and by residue R234 of a symmetry-related enzyme molecule on the non-reducing end. The ability of BlCel5B to cleave C5 into glucose and C4 molecules in solution was demonstrated by enzymatic product profile mass spectrometry analysis (Fig. 2A). The C4 oligomer in the BlCel5B binding site is coordinated by hydrogen bonds to residues N36, H113, H114, N158, W301, and N303 and by a CH-π interaction with residue W47 (Fig. 1D). These residues belong to the CD and are conserved in the GH5 family.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10667</offset><text>BlCel5B enzymatic activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10694</offset><text>BlCel5B exhibits optimum activity toward carboxymethylcellulose (CMC; 8.7 U/mg) at a pH of 4.0 and 55 °C and retains approximately half of its maximum activity at 80 °C, demonstrating considerable thermal stability (Fig. 2B,C). BlCel5B is also active on β-glucan (34 U/mg), lichenan (17.8 U/mg) and xyloglucan (15.7 U/mg) substrates (Table 1), whereas no activity was detected on galactomannan, rye arabinoxylan, 1,4-β-mannan or the insoluble substrate Azo-Avicel. Kinetic parameters were calculated assuming Michaelis-Menten behavior with CMC as substrate: KM = 1.78 g L−1 and Vmax = 1.41 × 10−4 g s−1 mg protein−1 (Fig. 2D). Although BlCel5B is not a highly active enzyme against one specific substrate as compared to others GH5_4, it has the advantage of being active against different substrates with β-1,3 and/or β-1,4 glycosidic linkages.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11580</offset><text>To understand the importance of the ancillary modules for BlCel5B activity, enzymatic assays were carried out using four enzyme mutants: a CBM46 deletion (ΔCBM46) and an Ig-like + CBM46 deletion (ΔIg-CBM46) as well as point mutations of the CBM46 inner surface residues W479A and W481A. These mutants were expressed and purified as described for the wild-type enzyme. Strikingly, neither of the deletion variants exhibited detectable activity toward any of the substrates tested using full-length BlCel5B (Table 1), demonstrating that the Ig-like module and the CBM46 are essential for BlCel5B activity. Thermal shift assays were conducted to confirm structural stability of the mutants (Supplementary Fig. 1). All of the constructs showed similar melting temperatures: 62 °C for BlCel5B, 58 °C for BlCel5BΔCBM46, 56 °C for BlCel5BΔIg-CBM46, 65 °C for BlCel5BW479A and 59 °C for BlCel5BW479A, thus confirming their proper overall fold.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12539</offset><text>We also examined the function of the CBM46 inner surface residues W479 and W481 (Fig. 1A) in BlCel5B activity by performing enzymatic assays with W479A and W481A mutants. Both mutations reduced enzymatic activity toward all tested substrates (Table 1), with W481A having a stronger effect than W479A (~64% vs. 79% activity relative to wt BlCel5B using β-glucan and ~10% vs. 50% using CMC). This indicates that CBM46 must interact with the substrate via residues W479 and W481. However, since the BlCel5B crystal structure exhibits no close contact between these residues and the substrate, these results suggest the existence of large-amplitude interdomain motions that may enable direct interactions between CBM46 and the carbohydrate.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13279</offset><text>BlCelB5 dynamics and binding-site architecture</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13326</offset><text>Molecular dynamics (MD) simulations were performed to investigate the conformational mobility of BlCel5B. In the simulations of the crystal structure for BlCel5B bound to C4, the substrate dissociates from the protein within the first 100 ns of the simulation time (Supplementary Fig. 2A). This observation suggests that cellotetraose does not exhibit detectable affinity for this specific BlCel5B conformation in solution, as one might otherwise expect for a reaction product. No changes beyond local fluctuations were observed in any of the three BlCel5B domains within the time scale of these runs (400 ns; Supplementary Fig. 2B). However, the CBM46 and Ig-like domains did exhibit rigid body-like motions relative to the CD, with rmsd values around 2.3 Å and 1.8 Å, respectively, suggesting that BlCel5B may execute large-amplitude interdomain motions over longer time scales (Supplementary Fig. 2B,C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14243</offset><text>Accordingly, simulations were then performed using accelerated molecular dynamics (aMD) techniques to probe BlCel5B interdomain motions. aMD enhances conformational sampling by raising the basins of the dihedral potential energy surface without affecting the general form of the atomistic potential, thereby increasing transition rates between different local minima. aMD trajectories corresponding to more than 1.0 μs of conventional MD runs were generated. During these simulations, we observed occlusive conformations between CBM46 and CD that resulted in a rearrangement of the enzyme’s architecture around the active site (Video S1). Figure 3A shows BlCel5B in the crystallographic conformation (red) and in a selected configuration obtained with aMD (blue) in the absence of the substrate. Interdomain motions were gauged by the time evolution of the distance between the α carbons of residues I120 and E477 (represented as spheres in Fig. 3A), belonging to the CD and CBM46, respectively. Figure 3C shows that the I120-E477 distance (red curve) gradually decreases from ~35 Å to ~7 Å within the first half of the 1.0 μs aMD trajectory, indicating a transition between the semi-open (crystallographic) and occluded (aMD sampled) configurations. During the second half of the aMD simulation, the full-length enzyme remained in the closed conformation, with the CBM46 covering the carbohydrate-binding site. These results suggest that BlCel5B undergoes large-scale interdomain movements that enable interactions between CBM46 and the substrate bound to the CD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15823</offset><text>To study the interactions of BlCel5B with a non-hydrolyzed glucan chain, we built a model structure with a cellooctaose (C8) chain spanning the entire positive (+1 to +4) and negative (−4 to −1) subsites of the enzyme. Starting from the crystallographic BlCel5B conformation, the C8 molecule deviated significantly from the active site and assumed a non-productive binding mode (Supplementary Fig. 2D). This observation suggests that the open conformation of BlCel5B is not able to hold the substrate in a position suitable for hydrolysis (Supplementary Fig. 2E). However, after subjecting the BlCel5B-C8 complex to a 0.5 μs aMD simulation with harmonic restraints on the C8 chain to prevent it from deviating from the productive binding mode, the CBM46 readily closed over the CD and trapped the C8 chain in position for hydrolysis (Fig. 3B). In the presence of the substrate, CBM46 adopts a final conformation intermediate between the crystallographic structure and that observed in the substrate-free BlCel5B aMD simulations; this is illustrated by the I120-E477 distance, which stabilizes near 20 Å in the closed configuration that traps the C8 molecule (in contrast to ~7 Å for substrate-free BlCel5B) (Fig. 3C). This BlCel5B-C8 configuration remains stable over an additional 500 ns of conventional MD simulation with no restraints (Fig. 3C cyan line, Supplementary Fig. 2E,F).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17220</offset><text>A closer inspection of the productive binding mode obtained from these extensive simulations reveals that the CBM46 tryptophan residues W479 and W481 (along with CD tryptophan residues) play important roles in carbohydrate recognition and orientation by creating a tunnel-like topology along the BlCel5B binding cleft, as depicted in Fig. 3D. Together, these results indicate that CBM46 is a key component of the catalytic active complex, providing an explanation as to why CBM46 is essential for the enzymatic activity of BlCel5B.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17752</offset><text>To enable substantially longer time scales compared to atomistic simulations, we further explored the dynamics of BlCel5B using coarse-grained MD (CG-MD) simulations. We performed three independent ~120 μs CG-MD simulations, for a total of approximately 360 μs of sampling. The distance between the α carbons of two residues centrally positioned in the CD and CBM46 (Fig. 4A) was monitored, and the results shown in Fig. 4B indicate that the wide-amplitude events described above frequently appear in this time scale. The computed distance distribution depicted in Fig. 4C indicates three main conformational states ranging from (I) closed conformations similar to those encountered in the substrate-free aMD simulations, in which CBM46 interacts with the CD to shape the substrate binding site, to (II) semi-open conformations similar to the crystallographic structure, and (III) extended BlCel5B conformations in which the CD and CBM46 are even further apart than in the crystal structure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18751</offset><text>BlCel5B conformers fit the SAXS envelope</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18792</offset><text>SAXS experiments were conducted to assess BlCel5B conformational states in solution, and the results revealed the enzyme in its monomeric form, with average values of Rg = 27.17 Å and Dmax = 87.59 Å (Supplementary Table 2). The ab initio dummy atom model (DAM) demonstrated that the SAXS-derived BlCel5B molecular envelope could not be single-handedly filled by any of the main conformational states encountered in the simulations (Fig. 4D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19248</offset><text>It is known that a Kratky plot exhibits a peak with an elevated baseline at high q for a monodisperse system composed of multi-domain particles with flexible extensions. Indeed, an elevation of the baseline toward a hyperbolic-like curve was observed for BlCel5B, indicating a considerable degree of molecular mobility in solution (Supplementary Fig. 3). Thus, the conformational heterogeneity of the enzyme can be decomposed in structural terms as a combination of conformational states identified in our crystallographic and MD studies. We found that the SAXS envelope can be well represented by considering the superimposition of three different representative molecular conformations of BlCel5B (Fig. 4E): a closed or CBM46/CD-occluded conformation extracted from the simulations with a relative weight of 26%, a semi-open conformation represented by the crystal structure corresponding to 40%, and an extended conformation based on simulations that is responsible for 34% of the SAXS envelope. The resulting average scattering curve from this model fits the experimental protein scattering intensity, with χ = 1.89 (Supplementary Fig. 3).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20397</offset><text>GH5_4 phylogenetic analysis</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20425</offset><text>To date, there are 427 sequences classified as subfamily 4 members in the CAZy database. After the exclusion of partial sequences and the suppression of highly identical members (higher than 90% identity), 144 sequences containing between 277 and 400 residues were aligned and used to construct a phylogenetic tree (Supplementary Fig. 4A). According to PFAM database conserved domain classification, 128 GH5 enzymes have an architecture consisting of an N-terminal catalytic module, a CBM_X2 module and an unknown module of approximately 100 residues at the C-terminus (Supplementary Fig. 4B). Of these, 12 enzymes have an additional CBM1, and 5 have a CBM2 at the N-terminal region. Based on this PFAM architecture and CAZy subfamily classification, all the 144 enzymes (including BlCel5B) belong to the GH5_4 subfamily and group together in the same branch of the phylogenetic tree, evidencing a common ancestor. These results support the hypothesis that the enzymes may employ the same mechanism by which ligand binding is mediated by an extensive conformational breathing of the enzyme that involves the large-scale movement of CBM46 around the Ig-like module (CBM_X2) as a structural hinge.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>21621</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>21632</offset><text>Growing interest in biotechnological applications of enzymes exhibiting activity toward lignocellulosic biomass has sparked efforts in the discovery and development of novel enzymes, as well as the search for a deeper understanding of their mechanisms of action. Here, we elucidate the trimodular molecular architecture of the full-length BlCel5B, a member of the GH5_4 subfamily, for which large-scale conformational dynamics appears to play a central role in its enzymatic activity. Full-length BlCel5B is active on both cellulosic and hemicellulosic substrates and auxiliary modules are crucial for its activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>22248</offset><text>Most carbohydrate-active enzymes are modular and consist of a catalytic domain appended to one or more separate AMs. AMs, such as CBMs, typically recognize carbohydrates and target their cognate catalytic domains toward the substrate. Because the structural analysis of the protein is challenging if the linkers connecting the structural subunits of the enzyme are long and flexible, the standard approach is to study the domains separately. In this work, a combination of protein crystallography, computational molecular dynamics, and SAXS analyses enabled the identification of a new conformational selection-based molecular mechanism that involves GH5 catalytic domain and two AMs in full-length BlCel5B. We observed that the BlCel5B distal CBM46 is directly involved in shaping the local architecture of the substrate-binding site. Although the CD alone appears unable to bind the substrate for catalysis, the AMs exhibit open-close motions that allow the substrate to be captured in a suitable position for hydrolysis. Here, we advocate that large-amplitude motions of AMs are crucial for assembling the enzyme into its active conformation, highlighting a new function of CBMs. This mechanism of substrate binding closely resembles the extended conformational selection model, with the induced-fit mechanism of reaction as its limiting case. To the best of our knowledge, this enzymatic mechanism has not been proposed previously for any GH.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23695</offset><text>The CD binding site of BlCel5B is open and relatively flat and is thus barely able to properly hold the substrate in position for catalysis without assistance from the CBM46. In contrast, other GH5s belonging to subfamily 4 listed in the Protein Data Bank exhibit a deep binding cleft or tunnel that can effectively entrap the substrate for catalysis (Fig. 5). Due to the marked interdomain conformational rearrangement observed in our simulations, the CBM46 generates a confined binding site in BlCel5B that resembles the binding site architecture of the other GH5 enzymes that lack AMs. Thus, BlCel5B appears to have adopted a strategy of CBM46-mediated interactions for proper functioning. Although the homologous BhCel5B has the same domain architecture of BlCel5B and belongs to the same subfamily (a comparison of the sequence and structure of BlCel5B and BhCel5B is presented in Supplementary Fig. 5), its binding site exhibits important differences that may impact the catalytic mechanism. The BhCel5B binding site is V-shaped and deeper than the BlCel5B binding site (Figs 5 and 6). This is due to the loop between residues F177 and R185 from BhCel5B (absent in the BlCel5B), which contains residue W181 that forms part of the binding cleft (Fig. 6). Consistently, although BhCel5B CBM46 is important for β-1,3-1,4-glucan hydrolysis (BhCel5B is about 60-fold less active without CBM46), the truncated enzyme is completely active against xyloglucan, suggesting that the CBM46, in this case, is necessary for the binding to specific substrates. A closer inspection of results of the phylogenetic analysis, more specifically of the clade composed by GH5_4 enzymes with trimodular architecture (Supplementary Fig. 4C), reveals subclades whose main characteristic is the varying length of the loop located between residues 161 and 163 (BlCel5B residue numbering). Therefore, our results show that BlCel5B represents a smaller group of enzymes that are completely dependent on its AMs for hydrolysis of plant cell wall polysaccharides, and that the underlying mechanism may rely on large-scale interdomain motions.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25816</offset><text>The amino acid sequence of the BlCel5B Ig-like module is recognized by BLASTP as belonging to CBM_X2, a poorly described group that has been compared with CBM-like accessory modules without a defined function. Despite the similarity of BlCel5B Ig-like module to CBMs, it lacks an identifiable aromatic residue-rich carbohydrate-binding site. Nonetheless, according to our results, the Ig-like module seems to play an important function as a structural hinge, dynamically holding the CBM46 and CD in positions that are appropriate for enzymatic activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26370</offset><text>Based on the results of our crystallographic, computer simulation, and SAXS structural analyses, as well as site-directed mutagenesis and activity assays, we propose a molecular mechanism for BlCel5B substrate binding, which might apply to other GH5_4 subfamily enzymes that share this tri-modular architecture. BlCel5B can be found in several different conformational states ranging from CBM46/CD closed (or occluded) to extended conformations (Fig. 7). In extended configurations, the substrate may dock at the shallow substrate binding site of CD in one of the semi-closed conformations of the enzyme; however, its binding is properly stabilized for hydrolysis only with the aid of induced-fit repositioning mediated by CBM46. After cleavage, the intrinsic dynamics of BlCel5B would eventually allow the opening of the active site for product release. The proposed mechanism is consistent with our mutagenesis and enzymatic activity assays, which show that the Ig-like module and CBM46 are indispensable for BlCel5B catalytic activity and, together with the CD, form the unique catalytic domain of the enzyme. These experiments reveal a novel function for CBMs in which they are intimately involved in the assembly of the active site and catalytic process. Computer simulations suggest that large-scale motions of the CBM46 and Ig-like domains mediate conformational selection and final induced-fit adjustments to trap the substrate at the active site and promote hydrolysis. SAXS data support the modeling results, providing compelling evidence for highly mobile domains in solution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>27958</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27966</offset><text>Cloning, Expression and Purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28003</offset><text>The gene encoding BlCel5B (GenBank: AAU23417.1) was amplified from Bacillus licheniformis genomic DNA (ATCC 14580) without the predicted signal peptide sequence (nucleotides 1 to 81) using the primers Blcel5B_Fw and Blcel5B_Rv (Supplementary Table 3). The fragment was cloned into the expression vector pETTRXA-1a/LIC by ligation-independent cloning (LIC), as described elsewhere.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28384</offset><text>The same method was used for construction of domain deletions. For Ig-like + CBM46 deletion, Δ(Ig-CBM46), the fragment encoding the CD (nucleotides 82 to 1086) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1087-1683_Rv. For CBM46 deletion, ΔCBM46, the fragment encoding the CD + Ig-like (nucleotides 82 to 1377) was amplified using the primers Blcel5B_Fw and Blcel5BΔ1378-1683_Rv (Supplementary Table 3). Both fragments were cloned into pETTRXA-1a/LIC.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28862</offset><text>The wt protein BlCel5B, mutated proteins and AM deletions were expressed in E. coli Rosetta2 (DE3) strain. The cells were grown at 37 °C and 150 RPM in Luria Bertani Broth medium supplemented with 50 μg/mL kanamycin to an A600 of 1.5–2.0, after which the temperature was reduced to 20 °C and protein expression was induced with 1 mM IPTG for 6 h.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29224</offset><text>The extract was then loaded onto a NiNTA resin (Qiagen) equilibrated with a washing buffer (5 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). Non-absorbed material was washed with ten times column volume with washing buffer and the purified protein was eluted with 200 mM imidazole, 100 mM NaCl, 50 mM Tris-HCl at pH = 7.0. His6 tag was removed by overnight digestion with TEV (Tobacco Etch Virus) at 4 °C, and untagged protein was purified by gel filtration through a HiLoad 16/60 Superdex 200 column in buffer containing 50 mM NaCl, 25 mM Tris-HCl at pH 7.0.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29812</offset><text>Site-directed Mutagenesis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29838</offset><text>The BlCel5B point mutations W479A and W481A were obtained by the inverse PCR method of site-directed mutagenesis. Phusion® “High-Fidelity” DNA polymerase (NEB, USA) was used for amplifications with the plasmid pETTRXA-1a/LIC-Blcel5B as a template. Mutagenic primers Blcel5BW479A_Fw/Rv and Blcel5BW481A_Fw/Rv (Supplementary Table 3) were generated by HTP-OligoDesigner tool (http://www.ifsc.usp.br/htpoligo/).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>30252</offset><text>Activity Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>30268</offset><text>Enzymatic activity assays were performed by a colorimetric method using the 3,5-dinitrosalicylic acid (DNS), with glucose being a standard for the calibration curves. Assays of optimal temperature and pH were performed in triplicate with 1% medium-viscosity CMC as the substrate. For optimal temperature, the reaction mixture containing 10 μL of enzyme at 0.1 mg/mL, 50 μL of 1% (w/v) CMC and 40 μL of 50 mM sodium citrate buffer (pH 5.0) was incubated at 30 to 80 °C for 15 min and stopped by adding 100 μL of DNS solution. After this, the mixture was incubated again for 5 min at 100 °C and the absorbance was measured at 540 nm with a spectrophotometer. For optimal pH determination, the same amount of enzyme and substrate were diluted in 40 mM acetate/borate/phosphate buffer (ABF) with different pH values ranging from 2.0 to 10.0. The reactions were carried out under the predetermined optimal temperature.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31212</offset><text>The substrate specificity of the enzyme was determind using rye arabinan, xyloglucan, β-glucan, galactomannan, lichenan, β-mannan, Azo-Avicel and CMC as substrates. The substrates were diluted in water to 1% (w/v), and the reaction mixture was composed of 10 mL of purified enzyme at a concentration of 0.1 mg/mL, 0.4 mL of 50 mM sodium citrate buffer at pH 5.0, and 0.5 mL of 1% (w/v) substrate aqueous solution. The reaction was incubated at 50 °C for 15 min, followed by treatment with DNS as mentioned above. Enzyme unit was defined as the amount of enzyme that produces 1.0 μM of glucose in one minute for each substrate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31859</offset><text>The kinetic parameters were determined by increasing concentrations of CMC. Reactions were performed in 50 mM sodium citrate buffer (pH = 4.0) at 50 °C, and measured by DNS method as well. Kinetic constants were determined by non-linear regression using OriginPro 8.0.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32137</offset><text>Thermal Shift Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32158</offset><text>The thermal denaturation assays were performed using a Real Time PCR Machine (Stratagene Mx3005P) as described by Dupeux and co-workers. Briefly, the enzymes were diluted to 10 μM in 50 mM sodium citrate buffer (pH = 4.0) containing 1x SYPRO orange dye (Thermo Fisher Scientific). The fluorescence emission of the probe was monitored (excitation and emission at 492 and 516 nm, respectively) varying the temperature between 25 and 75 °C with the rate of 1 °C/min.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32639</offset><text>Cellopentaose Cleavage Experiment</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32673</offset><text>The full-length BlCel5B and AM deletion constructs were tested for product formation from cellopentaose. Cellopentaose (1.0 mM) was incubated with 25 μg of purified enzyme in 10 mM ammonium bicarbonate buffer (pH 7.0) in a total volume of 50 μL. The reaction was incubated for 90 min at 50 °C and then stopped by treatment at 100 °C for 5 min. After centrifugation for 10 min at 16,000 g the samples were subjected to MALDI/TOF-MS. Samples were supplemented with NaCl to a final concentration of 20 mM and 1 μL of the supernatant was co-crystallized with 1 μL 2,5-dihydroxybenzoic acid (10 mg/mL) in acetonitrile 30% and spotted on the target plate. The products were analyzed on Microflex LT MALDI-TOF (Bruker Daltonics) operating in positive ion mode. A single spectrum was obtained by averaging four independent spectra generated by 300 laser shots at 60% potency.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33575</offset><text>Crystallization, Data Collection, and Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33637</offset><text>After purification, BlCel5B was concentrated to 10 mg/mL for crystallization trials. Crystallization screens were set up using the sitting-drop vapor-diffusion method on a Cartesian PixSys 4200 (Genomic Solutions, United Kingdom) in a 96-well plate with drops formed by 100 nL protein solution plus 100 nL reservoir solution. The commercial kits Crystal Screen and Index (Hampton) were used as initial conditions. Crystals were grown at 18 °C between 3 and 7 days, and screened for diffraction.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34141</offset><text>Crystals were supplemented with cryoprotection solution, flash cooled in liquid nitrogen and diffraction data were collected at 100 K, at beamline ID23-1 (wavelength of 0.97 Å) from the European Synchrotron Radiation Facility (Grenoble, France). A crystal grown in condition containing 22.5% PEG 4000, 14% isopropanol and 0.1 M sodium citrate, pH 6.0, was selected to collect diffraction data to 1.7 Å resolution. The complex of the enzyme with substrate was obtained by crystal soaking with five times molar excess of cellopentaose for 24 hours. Diffraction data for the complexed enzyme were collected at 1.75 Å resolution.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34782</offset><text>Data were integrated with iMosflm and scaled with Aimless. The structure was solved by molecular replacement with Phaser using an endoglucanase from Clostridium cellulovoran (PDB code: 3NDY) as the search model. Coot was used for density fitting, and refinement was performed with PHENIX.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>35071</offset><text>Atomistic simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35093</offset><text>We took the BlCel5B structure complexed with cellotetraose as the starting configuration for the MD simulations. The missing residues were taken from the apo BlCel5B structure after structural alignment using the LovoAlign server. Hydrogen atoms were then added according to the protonation states determined at the optimum pH of 4.0 using the H + + server. The following residues were considered protonated: H55, H77, D89, E96, E103, H114, E129, E159, E197, D198, E202, H205, E208, D211, H220, E245, E248, E260, H278, H292, D306, E312, E371, E375, E476, H416, E477, E489, D497, and E524. The remaining protonatable residues were considered in the standard protonation state. The BlCel5B-cellotetraose complex was then immersed in a rectangular simulation box of dimensions such that a solvent layer at least 16 Å thick surrounded the protein. The simulation box, built with Packmol, also contained 0.10 M NaCl aqueous solution with excess counter ions to keep the system electrically neutral. The final system comprised approximately 85500 atoms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36152</offset><text>The simulations were performed using NAMD with the CHARMM force field and the TIP3P water model. Periodic boundary conditions were employed, using particle mesh Ewald to handle electrostatics and a 12-Å cutoff radius for truncating short-range potentials. Bonds involving hydrogen atoms were constrained at their equilibrium lengths and a time step of 2 fs was used to integrate the equations of motion. The simulations were carried out under constant pressure and temperature of 1 atm and 310 K, respectively, employing the Langevin barostat and thermostat.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36714</offset><text>Accelerated Molecular Dynamics</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36745</offset><text>In accelerated molecular dynamics, the trajectory is propagated on a modified potential aimed to enhance conformational sampling. Whenever the potential energy drops below a given threshold E, a boost ΔV(r) is applied, so that the escaping rates of local minima increase. When the potential energy gets over the threshold E, the system evolves on the original energy surface. This method has the advantage of conserving the general shape of the potential energy surface and of requiring no prior definition of reaction coordinates, so the system is allowed to explore freely its conformational space. Here, we restricted the energy boost only to the dihedral potential energy, as changes in torsion angles are the main source of conformational changes in proteins. The energy boost assumes the form of equation (1) that depends on the energy threshold E and on the parameter α – which modulates the shape of the potential energy surface where the boost is applied. We set the parameters E and α according to previous studies, which recommend that E equals the average dihedral energy obtained from a conventional MD simulation plus 4 kcal/mol times the number of residues, and α equals 0.8 kcal/mol times the number of residues. The average dihedral energy was 2275.5 kcal/mol and the BlCel5B has 516 residues, so we set E = 2275.5 + 4 × 516 = 4339.5 kcal/mol and α = 0.8 × 516 = 418.8 kcal/mol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38188</offset><text>Simulation procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38210</offset><text>BlCel5B-cellotetraose – Having built the system, we carried out the following steps for equilibration: (i) 1000 steps of energy minimization followed by 100 ps of MD simulation with all non-solvent heavy atoms fixed; (ii) same as (i), but with only the α carbons fixed; (iii) 5 ns of MD with all atoms free. After these preliminary steps, a trajectory lasting 400 ns was generated using conventional MD and then the aMD dihedral boost was applied for additional 1.0 μs. After 100 ns of conventional MD, the cellotetraose dissociated and the simulation began to represent the dynamics of unbound state of BlCel5B.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>38835</offset><text>BlCel5B-cellooctaose</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38856</offset><text>To build the cellooctaose chain, we extended the original cellotetraose chain in the crystal structure with 4 additional glucose residues spanning regions around the BlCel5B positive subsites. Then, we submitted the system to the following procedure: (i) 1000 steps of energy minimization followed by 1 ns of MD keeping all the non-solvent heavy atoms fixed, except the 4 modeled glucose residues of the cellooctaose chain; (ii) same as step (i), but with only the α carbons fixed; (iii) 1 ns of MD with only the non-modeled glucose residues fixed. We then performed a 200-ns-long MD with three harmonic potentials involving cellooctaose chain: first, between C3 atom (CHARMM atom names) of the second glucose residue from the cellooctaose non-reducing end and the CD2 atom of the W47 tryptophan residue; second, between the OH3 atom of the forth glucose residue from the cellooctaose non-reducing end and HE2 atom of the H113 histidine residue; and third, between the HE2 atom of the catalytic residue E159 and O4 glycosidic oxygen between the fourth and fifth glucose unit of the cellooctaose chain. After these preliminary relaxation steps, the harmonic potentials were removed and the trajectory was propagated by 400 ns using MD. To get a model of the BlCel5B-cellooctaose complex in the closed conformation, we took the configuration after 80 ns of the restrained 200-ns MD simulation as the starting point for a 500-ns-long restrained aMD simulation, in which the CBM46 moved towards the CD in the presence of the harmonically-restrained cellooctaose chain. After this procedure, we released the restraints and propagated the closed BlCel5B-cellooctaose complex for additional 500 ns of conventional, restraint-free MD simulation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40605</offset><text>Coarse-grained MD simulations</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40635</offset><text>The coarse-grained model was constructed from the minimized all-atom protein. We have used the domELNEDIN CG model for the protein. In this representation, an elastic network is used within each domain as a structural scaffold in order to maintain the overall shape of the protein, and a slightly modified version of MARTINI CG model describes the interactions involving beads not connected by harmonic springs.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41047</offset><text>The delimitation of each domain was quite clear considering the short linkers connecting them and the recognition of their structural patterns in databases. We assumed CD, Ig-like module, and CBM46 as consisted of residues 18–331, 332–430, and 431–533, respectively. Therefore, there were elastic network bonds only within these domains (domELNEDIN CG model in Supplementary Fig. 6A).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41438</offset><text>The protonation state of each residue bead in the protein was the same adopted in the atomistic simulations. The system was then solvated by 10000 standard MARTINI CG water beads, including 10% of antifreeze particles. Also, 58 chloride and 48 sodium ions were added for charge neutrality. The size of final system was 109 Å × 109 Å × 109 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41799</offset><text>Preliminary simulations were performed to test the elastic network (EN) parameters. We have tested six different ENs in 100 ns of simulations, using combinations of cut-off distance (Rc) of 8 Å and 9 Å with spring force constant (ks) of 500, 800 and 1000 kJ mol−1 nm−2. The time evolution of root mean square deviation relative to the crystal structure as well as the mobility profile of the protein in these simulations were compared to the correspondent data from a 100 ns atomistic simulation. From this procedure, the parameters Rc = 9 Å and ks = 500 kJ mol−1 nm−2 resulted in the best match between atomistic and coarse-grained simulations (Supplementary Fig. 6B).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42500</offset><text>The coarse-grained simulations were carried out using GROMACS. Periodic boundary conditions were employed. Van der Waals interactions were shifted to zero in the range 0.9–1.2 nm, and the electrostatic interactions, in the range 0.0–1.2 nm. The simulations were performed in the isothermal-isobaric ensemble (NpT), employing the Berendsen thermostat and barostat for temperature and pressure control, respectively, with time constants τT = 0.5 ps and τp = 1.2 ps.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42982</offset><text>The CG simulations were carried out using the following protocol: the system was first minimized for 1000 steps using the steepest descent method. Then, it was submitted to a relaxation procedure comprising gradual increasing in time step or temperature. In the first stage of relaxation, the protein beads were restrained with a 1000 kJ mol−1 nm−2 force constant and a 50 ps simulation was carried out at 50 K, using the short time step of 1 fs. In the second stage, the time step was increased up to 5 ps lasting 1000 ps of simulation time. In the last stage of relaxation, all the system is released to move and it underwent a gradual increase in temperature, consisting on five segments of 100 ps at 50, 100, 150, 200 and 310 K. After achieving the desired temperature of 310 K, we performed three production simulations using 20-fs timestep. We have used a random number generator for assigning velocities to generate three independent simulations.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>43950</offset><text>In general, smoothing of the energy surface in CG model makes the time scales faster. A speed up factor of 4 is typically employed to rescale the time scale of MARTINI CG systems. Therefore, all CG simulations times described here and in the main text are effective times, i.e., 4× simulation time.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>44250</offset><text>Small Angle X-ray Scattering</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44279</offset><text>SAXS data were collected at the SAXS2 beamline of the Brazilian Synchrotron Light Laboratory-LNLS (Campinas, Brazil) on a bi-dimensional position sensitive CCD detector (MarResearch, USA) using the radiation wavelength 1.54 Å. The sample-detector distance of 1000 mm allowed covering the momentum transfer range 0.01 Å−1 < q < 0.35 Å−1 (q = 4πsin θ/λ, where 2θ is the scattering angle).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44697</offset><text>The protein samples were prepared in McIlvaine’s buffer at 50 mM, pH 5 and 20 °C. In each measurement, two successive frames of 300s were recorded for each sample at 1 and 2 mg/mL to monitor radiation damage. The patterns were integrated using the FIT2D program. The comparative analysis for each scattering curve at 1 and 2 mg/mL of BlCel5B (data not shown), as well as the radius of gyration values (Rg), indicated that concentration and aggregation effects did not exist. The linearity of the Guinier plot indicated that the preparation was monodisperse.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45266</offset><text>The radius of gyration of the molecules (Rg) was estimated by two methods, using the Guinier equation-I(q) = I(0).exp[(−q2.Rg2)/3], q.Rg < 1.3- and also with the inverse Fourier transform in GNOM. The same program was used to obtain the distance distribution function P(r) and the maximum diameter Dmax. Ten independent dummy atom models (DAMs) were restored by the ab initio proceeding implemented in DAMMIN package. The best model, selected using normalized spatial discrepancy parameter computed by DAMAVER program, was superimposed on the crystallographic model with the SUPCOMB.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45860</offset><text>Then, based on the enzyme conformations reported by MD and protein crystallography, the computed X-ray scattering profile was fitted to a given experimental SAXS data by minimizing the χ function in the FOXS program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46079</offset><text>To assess the inter-domain information, the contribution of individual conformer and the flexibility of BlCel5B, we proceed in two approaches. First, the theoretical profiles and experimental data comparison was performed to infer the best-fit conformation of the ensemble-based analysis by the ensemble optimization method – EOM, which assumes coexistence of a range of conformations in solution for which an average scattering intensity fits the experimental SAXS data; all models were generated with the three individual domains (Ig-like, CBM46, and CD) free to randomly move in order to cover the entire conformational space. The second approach was based on a fractional volume calculation from three conformation members extracted from the MD simulations, each with a distinct scatter curve. OLIGOMER provided solution of a system of linear equations between the experimental and generated conformations by MD.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46998</offset><text>The simulated scattering curves from the MD and crystallographic models were obtained using the CRYSOL.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47102</offset><text>Phylogenetic assignment</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47126</offset><text>Sequences for all GH5 members, in which only the catalytic domain were considered, were downloaded from PFAM database and their classification into subfamilies were obtained within the CAZy database. The sequences belonging to subfamily 4 were selected and those that had over 90% identity or represented partial coverage were rejected. Based on their multiple sequence alignment, the phylogenetic tree was constructed using the maximum likelihood method implemented in the MEGA program version 6.06. One hundred Bootstrap replications were performed to examine the reliability of the phylogenetic tree.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>47730</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47753</offset><text>How to cite this article: Liberato, M. V. et al. Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism. Sci. 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R.L.S., E.T.P. and M.S.S. designed the computer simulations; R.L.S. performed Accelerated Molecular Dynamics; E.T.P. performed Coarse-grained MD Simulations; M.V.L., R.L.S., M.S.S. and I.P. wrote the manuscript with the input from all the other authors; M.S.S. and I.P. supervised the project.</text></passage><passage><infon key="file">srep23473-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>53941</offset><text>Crystal models of BlCel5B.</text></passage><passage><infon key="file">srep23473-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53968</offset><text>Complete structure is shown as a cartoon illustration in (a) and a van der Waals surface in (b). The CD module (red) has a typical TIM-barrel fold, and its substrate-binding site is adjacent to CBM46 (blue). Despite the proximity of the binding site in the crystallographic model, the CBM46 residues W479 and W481 are distant from the substrate cellotetraose (yellow). The Ig-like domain (green) has a lateral position, serving as a connector between the CD and CBM46. (c) A superposition of the Ig-like domain and CBM46 illustrates their structural similarity, with most of the structural differences present in the loop highlighted by a red circle. (d) Cellotetraose occupies subsites -1 to -3 and is primarily coordinated by the residues represented in gray.</text></passage><passage><infon key="file">srep23473-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54730</offset><text>BlCel5B enzymatic activity characterization.</text></passage><passage><infon key="file">srep23473-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54775</offset><text>(a) MALDI/TOF-MS spectra of the products released after incubation of BlCel5B and its two deletion constructs (ΔCBM46 and ΔIg-CBM46) with the substrate cellopentaose (C5). The first three spectra show the substrate, enzyme and buffer controls. The forth spectrum reveals that full length BlCel5B is capable of enzymatic hydrolysis of C5 into smaller oligosaccharides such as C4, C3 and C2. The last two spectra show that the C-terminal deletions eliminate the enzyme activity. BlCel5B activities on CMC as functions of pH and temperature are shown in (b) and (c), respectively. The enzyme exhibits optimal pH of 4.0 and optimal temperature of 55 °C, retaining about 50% of its activity at 80 °C. (d) Michaelis-Menten curve using CMC as a substrate.</text></passage><passage><infon key="file">srep23473-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>55532</offset><text>Open-close transitions of BlCel5B.</text></passage><passage><infon key="file">srep23473-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>55567</offset><text>(a) BlCel5B in the absence of substrate and (b) in the presence of cellooctaose, as observed in our aMD simulations. The distance between the α carbon of residues I120 (CD) and E477 (CBM46), illustrated as spheres in (a), is plotted in (c), revealing a transition by the decrease in the distance from 40 Å to 7 Å (substrate-free) or 20 Å (in presence of cellooctaose). For the substrate-free enzyme, the red line refers to a 1 μs-long aMD; for the BlCel5B-cellooctaose complex, the first 500 ns refers to aMD (in blue) and the second 500 ns to conventional MD (in turquoise). (d) A snapshot of the BlCel5B-cellooctaose complex, highlighting the tryptophan residues that interact with the glucan chain in subsites −4 to +4. Residues W479 and W481 belong to CBM46 and only become available for substrate interactions in the closed configuration of BlCel5B.</text></passage><passage><infon key="file">srep23473-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>56441</offset><text>Large-scale movements of BlCel5B modules and superposition of their representative conformations with the SAXS envelope.</text></passage><passage><infon key="file">srep23473-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>56562</offset><text>(a) BlCel5B structure showing the distance between the backbone beads of residues I120 and E477, which are centrally located in CD and CBM46, respectively, as a metric for the relative disposition between the two domains. (b) Time history of the I120-E477 distance computed using CG-MD simulations. Different colors separated by vertical lines correspond to independent simulations of approximately 120 μs. (c) The distance distribution indicates three major peaks: closed or occluded CBM46/CD conformations (I); semi-open (II), which is similar to the crystallographic structure; and extended conformers (III). (d) Superimposition of the three representative molecular conformations of BlCel5B with the SAXS model. (e) Average structures obtained from the simulation segments corresponding to population groups I-III, which are individually superposed on the SAXS envelope.</text></passage><passage><infon key="file">srep23473-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>57440</offset><text>Comparison of the binding site shape of GH5_4 enzymes available on the Protein Data Bank.</text></passage><passage><infon key="file">srep23473-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>57530</offset><text>(a) BlCel5B in the crystallographic and closed configuration; (b) Bacillus halodurans Cel5B (BhCel5B) (PDB id: 4V2X) (c) Piromyces rhizinflata GH5 endoglucanase (PDB id: 3AYR); (d) Clostridium cellulolyticum GH5 endoglucanase (PDB id: 1EDG); (e) Clostridium cellulovorans GH5 endoglucanase (PDB id: 3NDY); (f) Bacteroides ovatus GH5 xyloglucanase (PDB id: 3ZMR); (g) Paenibacillus pabuli GH5 xyloglucanase (PDB id: 2JEP); (h) Prevotella bryantii GH5 endoglucanase (PDB id: 3VDH); (i) Ruminiclostridium thermocellum multifunctional GH5 cellulase, xylanase and mannase (PDB id: 4IM4); (j) Bacteroidetes bacterium AC2a endocellulase (PDB id: 4YHE).</text></passage><passage><infon key="file">srep23473-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>58176</offset><text>Comparison of the binding cleft of the BlCel5B and BhCel5B.</text></passage><passage><infon key="file">srep23473-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>58236</offset><text>The main difference between BlCel5B and BhCel5B is that the latter exhibits a deeper cleft due to the presence of residue W181 in the loop between F177 and R185. We conjecture that this difference in the binding site architecture relates to the importance that the CBM46 plays in the BlCel5B enzymatic mechanism.</text></passage><passage><infon key="file">srep23473-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>58549</offset><text>Proposed molecular mechanism of BlCel5B conformational selection.</text></passage><passage><infon key="file">srep23473-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>58615</offset><text>As suggested by the simulations and SAXS data, BlCel5B spans multiple conformations ranging from closed to extended CBM46/CD states. In a given open state, the substrate may reach the active site and become entrapped by the capping of CBM46 onto CD and induced-fit conformational adjustments. After hydrolysis, the reaction product is released to yield apo-BlCel5B, which becomes ready for a new cycle.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>59018</offset><text>Activity of BlCel5B constructs against tested substrates.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th rowspan="2" align="left" valign="bottom" charoff="50">Substrate (1%)</th><th colspan="5" align="center" valign="top" charoff="50">Relative Activity (%)</th></tr><tr><th align="center" valign="top" charoff="50">WT<xref ref-type="fn" rid="t1-fn1">*</xref></th><th align="center" valign="top" charoff="50">W479A</th><th align="center" valign="top" charoff="50">W481A</th><th align="center" valign="top" charoff="50">ΔCBM46</th><th align="center" valign="top" charoff="50">ΔIg-CBM46</th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50">β-glucan</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">79.1</td><td align="center" valign="top" charoff="50">63.6</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">CMC</td><td align="center" valign="top" charoff="50">25.5</td><td align="center" valign="top" charoff="50">12.2</td><td align="center" valign="top" charoff="50">2.4</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Lichenan</td><td align="center" valign="top" charoff="50">52.4</td><td align="center" valign="top" charoff="50">41</td><td align="center" valign="top" charoff="50">28.6</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Xyloglucan</td><td align="center" valign="top" charoff="50">45.2</td><td align="center" valign="top" charoff="50">41.2</td><td align="center" valign="top" charoff="50">30.8</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Azo-Avicel</td><td align="center" valign="top" charoff="50">nd<xref ref-type="fn" rid="t1-fn2">**</xref></td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Arabinoxylan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">Galactomannan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr><tr><td align="left" valign="top" charoff="50">1,4-β-mannan</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td><td align="center" valign="top" charoff="50">nd</td></tr></tbody></table>
</infon><offset>59076</offset><text>Substrate (1%) Relative Activity (%) WT* W479A W481A ΔCBM46 ΔIg-CBM46 β-glucan 100 79.1 63.6 nd nd CMC 25.5 12.2 2.4 nd nd Lichenan 52.4 41 28.6 nd nd Xyloglucan 45.2 41.2 30.8 nd nd Azo-Avicel nd** nd nd nd nd Arabinoxylan nd nd nd nd nd Galactomannan nd nd nd nd nd 1,4-β-mannan nd nd nd nd nd </text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>59407</offset><text>*WT = wild type.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>59428</offset><text>**nd = not detected.</text></passage></document></collection>
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<collection><source>PMC</source><date>20230107</date><key>pmc.key</key><document><id>4980666</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep31500</infon><infon key="article-id_pii">srep31500</infon><infon key="article-id_pmc">4980666</infon><infon key="article-id_pmid">27510745</infon><infon key="elocation-id">31500</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Carcelli;given-names:Mauro</infon><infon key="name_1">surname:Rogolino;given-names:Dominga</infon><infon key="name_2">surname:Gatti;given-names:Anna</infon><infon key="name_3">surname:De Luca;given-names:Laura</infon><infon key="name_4">surname:Sechi;given-names:Mario</infon><infon key="name_5">surname:Kumar;given-names:Gyanendra</infon><infon key="name_6">surname:White;given-names:Stephen W.</infon><infon key="name_7">surname:Stevaert;given-names:Annelies</infon><infon key="name_8">surname:Naesens;given-names:Lieve</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>97</offset><text>Influenza virus PA endonuclease has recently emerged as an attractive target for the development of novel antiviral therapeutics. This is an enzyme with divalent metal ion(s) (Mg2+ or Mn2+) in its catalytic site: chelation of these metal cofactors is an attractive strategy to inhibit enzymatic activity. Here we report the activity of a series of N-acylhydrazones in an enzymatic assay with PA-Nter endonuclease, as well as in cell-based influenza vRNP reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity in the low micromolar concentration range and good selectivity. Computational docking studies are carried on to investigate the key features that determine inhibition of the endonuclease enzyme by N-acylhydrazones. Moreover, we here describe the crystal structure of PA-Nter in complex with one of the most active inhibitors, revealing its interactions within the protein’s active site.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1057</offset><text>Influenza virus is an enveloped virus with a segmented negative-oriented single-stranded RNA genome, belonging to the Orthomyxoviridae. Seasonal influenza A and B viruses affect each year approximately 5–10% of the adult and 20–30% of the paediatric population, and there is a permanent risk of sudden influenza pandemics, such as the notorious ‘Spanish flu’ in 1918 and the swine-origin H1N1 pandemic in 2009. Two classes of anti-influenza virus drugs are available, acting on the viral M2 ion-channel (amantadine and rimantadine) or on the viral neuraminidase (zanamivir and oseltamivir). The M2 inhibitors have limited clinical utility due to their central nervous system side effects and widespread resistance, as in the case of the 2009 pandemic H1N1 virus; resistance is also a growing concern for oseltamivir. Therefore, there is an urgent need for new antiviral drugs with an entirely different mode of action.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1984</offset><text>The influenza virus polymerase complex is composed of three subunits: PB1, PB2 and PA. The PA subunit performs the ‘cap-snatching’ endonuclease reaction, the PB2 subunit is responsible for initial binding of the capped RNAs, while the actual RNA synthesis is performed by the PB1 protein.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2277</offset><text>Given its crucial role in the viral life cycle, the influenza virus polymerase is widely recognized as a superior target for antiviral drug development and, in particular, inhibition of the PA endonuclease has deserved much attention in recent years.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2528</offset><text>The endonuclease catalytic site resides in the N-terminal domain of PA (PA-Nter; residues 1~195). It comprises a histidine (His41) and a cluster of three strictly conserved acidic residues (Glu80, Asp108, Glu119), which coordinate (together with Ile120) one, two, or three manganese or magnesium ions. The two-metal-ion model is consistent with numerous biochemical findings. Since the intracellular concentration of Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant. A controversy about number and type of metal ions exists also for the active site of HIV-1 integrase. HIV-1 integrase inhibitors are a paradigm for the innovative drug concept that is based on coordination with the metal cofactor(s) of viral enzymes: similarly, several PA-binding agents with metal-chelating properties have been identified as influenza endonuclease inhibitors (Fig. 1), including 2,4-dioxobutanoic acid derivatives, flutimide and its derivatives, 2-hydroxyphenyl amide derivatives, as well as tetramic acids, 5-hydroxypyrimidin-4-one derivatives, marchantins and green tea catechins, like epigallocatechin-3-gallate (EGCG, Fig. 1).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3693</offset><text>In recent years, we focused our research on chemical scaffolds that are able to chelate metal ions of PA-Nter, resulting in inhibition of influenza virus replication. N-acylhydrazones represent an appealing class of chelating ligands with a broad spectrum of biological activities, such as activity against HIV, hepatitis A, vaccinia and influenza virus. In the present work, we report the biological activity of a series of N-acylhydrazones (Fig. 2), as determined in an enzymatic assay with PA-Nter endonuclease as well as in cell-based influenza viral ribonucleoprotein (vRNP) reconstitution and virus yield assays. Several N-acylhydrazones were found to have promising anti-influenza activity with 50% effective concentration values (EC50) in the range of 3–20 μM and good selectivity (Table 1 and Fig. 3). Computational docking studies of two candidate ligands in the PA-Nter active site gave information about the features that could determine inhibition of endonuclease activity. Moreover, we describe the X-ray crystal structure of PA-Nter in complex with one of the most active inhibitors.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4797</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4820</offset><text>Chemistry</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4830</offset><text>N-acylhydrazones 1–27 (Fig. 2) were prepared in high yields by following literature methods (Fig. 2A); they were characterized by spectroscopic tools, mass spectrometry and elemental analysis. Even if isomerism around the C = N bond is possible, 1–27 are present in the E form in solution, as evidenced by the chemical shift values of the HC = N and NH protons in the 1H-NMR spectrum. Exceptions are represented by the alkyl-derivatives 3 and 4 (2:1 and 5:3 E:Z ratio, respectively).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5326</offset><text>If R’ (Fig. 2A) is a 2-hydroxy substituted phenyl ring, the corresponding acylhydrazones can coordinate one or, depending on denticity, two metal centers (modes A and B in Fig. 4). Starting from N’-(2,3-dihydroxybenzylidene)-semicarbazide (1) and its methoxy-analogue (2), we modified the acylhydrazonic substituent R” (3–8, 18, 19, Fig. 2A). In 18 and 19, also the gallic moiety can be involved in the chelation of the metal cofactors (mode C, Fig. 4). In order to investigate the role of hydroxyl substituents 9–11, 13–17, 20–23 and 27 were also synthesized. Compound 12 was synthesized in order to confirm the crucial influence of the gallic moiety. Finally, 26 was here considered, because it is an inhibitor of HIV RNase H, another enzyme with two magnesium ions in its active site.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6128</offset><text>Since the inhibitory activity of the N-acylhydrazones could be related to chelation of the divalent metal cofactor(s) in the influenza PA-Nter active site, we investigated the coordination properties of one model ligand (i.e. 19, H2L) towards Mg2+. Different reaction conditions were used (1:1 and 1:2 metal to ligand ratio, up to 4 equivalents of triethylamine), but in any case the same chemical species Mg(HL)2∙4H2O was recovered and conveniently characterized. The use of a coordinating solvent as d6-DMSO causes partial decoordination of the ligand, but the 1H-NMR spectrum in MeOD, instead, shows only the signals attributable to the complex. In the 13C-NMR spectrum, the signal of the C = O quaternary carbon is practically unaffected by complexation, suggesting that the C = O group is weakly involved in the coordination to the metal ion. This is confirmed, in the IR spectrum, by the shift of about 20 cm−1 of the C = O absorption, while a shift of 30–50 cm−1 is expected when the carbonylic oxygen is tightly bound to the metal ion. ESI-mass spectra and elemental analysis confirmed the formula Mg(HL)2∙4H2O.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7274</offset><text>The interaction between the N-acylhydrazone ligands and the magnesium cation was investigated also by means of UV-visible spectroscopy (UV-visible titrations of 23 and 19 with increasing amount of Mg(CH3COO)2 are shown in Figure S1). The spectrum of 19 includes a band at 313 nm assignable to n-π* transitions of the C = N and C = O groups. By adding increasing equivalents of Mg(CH3COO)2, the absorption around 400 nm increases, and a new band appears with a maximum at 397 nm. The opposite trend is observed in the range 300–350 nm, where an isosbestic point is present close to 335 nm. When the same experiment was performed with 23, a different behavior was observed. Increasing concentration of Mg2+, in fact, caused a diminution in the maximum absorption, an isosbestic point is visible at about 345 nm, but a new band at 400 nm does not appear. Ligands 19 and 23 coordinate the Mg2+ ions in different ways: 19 chelates the metal ion by using the deprotonated salicyl oxygen and the iminic nitrogen, while for 23, the gallic moiety is supposed to be involved (Fig. 4A,B versus C), leading to different, less extensive, modifications of the UV spectrum. These results will be revisited during the discussion of the biological activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8540</offset><text>Inhibition of the PA-Nter enzyme</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8573</offset><text>All the compounds were tested for their ability to inhibit the influenza endonuclease in an enzymatic plasmid-based assay with recombinant PA-Nter, as well as in cell-based influenza methods (i.e. virus yield and vRNP reconstitution assays). The results are shown in Table 1 and summarized in Fig. 3 to visualize the structure-activity relationships; Figure S2 shows the dose-response curves for three representative compounds (i.e. 10, 13 and 23) in either the PA-enzyme or vRNP reconstitution assay.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9075</offset><text>The moderate activity (IC50 = 24 μM) of N’-2,3-dihydroxybenzylidene semicarbazide (1) was completely lost when the NH2 moiety was replaced by a hydrophobic heptyl chain (3), but it is less affected when a phenyl or a 2-hydroxyphenyl is present (5 and 7, IC50 = 84 and 54 μM, respectively). When the hydroxyl in position 3 on R1 (2,3-dihydroxybenzylidene) was replaced by a methoxy group (2-hydroxy-3-methoxybenzylidene), the activity disappeared (compounds 2, 4, 6 and 8). The activity is unaffected (IC50 values ranging from 45 to 75 μM) when going from two hydroxyls in R1 (7) to compounds with three hydroxyls (i.e. 9, 10 and 11). Similarly, 11 (R1 = 3,4,5-trihydroxyphenyl, R2 = 2-hydroxyphenyl) had comparable activity as 27 (R1 = 3,4,5-trihydroxyphenyl, R2 = NH2). Within the series carrying a 2-hydroxyphenyl R2 group, the activity of 11 is particularly intriguing. 11 does not have the possibility to chelate in a tridentate ONO fashion (mode A in Fig. 4), but it can coordinate two cations by means of its three OH groups in R1 (mode C, Fig. 4). Note that a similar chelating mode was observed in a crystal structure, solved by Cusack and coworkers, of PA-Nter endonuclease in complex with the inhibitor EGCG.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10329</offset><text>The PA-Nter inhibitory activity strongly depends on the number and position of hydroxyl substituents in R1 and R2: this is clearly highlighted by the data obtained with compounds 13–23, in which R2 is a 3,4,5-trihydroxyphenyl (gallic) group, the most active scaffold in our series. The analogue carrying an unsubstituted aromatic ring as R1 (compound 13) had moderate activity (IC50 = 69 μM). When one OH was added at position 2 of the R1 ring (14), the activity was lost. Adding a second OH substituent at position 5 resulted in strong activity (compound 15, IC50 = 9 μM); medium activity for a 3-OH (18; IC50 = 83 μM), and marginal activity when the second OH is at position 4 (17, IC50 ≥ 370 μM). The addition of a 3-methoxy group (19) abolished all inhibitory activity. This cannot be related to variations in the chelating features displayed by the R1 moiety, since compounds 14–19 all have, in theory, the capacity to chelate one metal ion through the ortho-OH and iminic nitrogen (mode A in Fig. 4). Moreover, compound 18 can, in principle, chelate the two M2+ ions in the active site according to mode B (Fig. 4), yet it (IC50 = 83 μM) has nine-fold lower activity than 15, that does not possess this two-metal chelating feature. Therefore, we hypothesized that the inhibitory activity of the series containing the gallic moiety is determined by: (i) the capacity of the moiety R2 to chelate two metal ions in the active site of the enzyme, according to mode C (Fig. 4); and (ii) the presence and position of one or more hydroxyl substituents in R1, which may possibly result in ligand-protein interactions (e.g. through hydrogen bonds). This assumption was supported by molecular docking calculations and X-ray analysis of inhibitor 23 in complex with PA-Nter (vide infra). At this point, change of the substituents in R1 represents the next logical step. Substitution of the 5-hydroxyl in 15 by a methoxy group (16) causes a dramatic drop in activity (IC50 = 9 and 454 μM for 15 and 16, respectively). When two or three OH groups are present in R1, their spatial disposition greatly affects the activity. In particular, all the compounds with a trihydroxylated phenyl group as R1 (i.e. 20, 21, 22 and 23) were able to inhibit PA-Nter quite potently. The lowest IC50 values were obtained for 21 and 23 (IC50 = 13 and 7 μM, respectively), which both have one of their three hydroxyl groups at position 5. The most active compound in this series was 23, which lacks the hydroxyl group at position 2 of R1, further confirming that this function is undesirable or even detrimental for inhibitory activity against PA-Nter, as already noticed above for 14.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13044</offset><text>Consistent with a crucial role of the R2 gallic moiety in metal chelation, the strong activity of 15 was completely lost in its 3,4,5-trimethoxy analogue 12. On the other hand, the R2 gallic containing compounds displayed moderate activity (IC50 values around 40 μM) when R1 was absent (i.e. the 3,4,5-trihydroxybenzohydrazide 28, Fig. 2), or composed of an extended ring system (26) or a pyrrole ring (25). Still lower activity was seen with the pyridine analogue 24. Evidently, the 3,4,5-trihydroxybenzyl moiety at R2 is fundamental but not sufficient to ensure potent PA-Nter endonuclease inhibition, since the interactions of R1 with the amino acid side chains of the protein appear crucial in modulating activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13766</offset><text>Inhibition of vRNP activity or virus replication in cells</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13824</offset><text>To determine the anti-influenza virus activity of compounds 1–28 in cell culture, we performed an influenza vRNP reconstitution assay in human embryonic kidney 293 T (HEK293T) cells, then subjected the active compounds (i.e. EC50 < 100 μM) to a virus yield assay in influenza virus-infected Madin-Darby canine kidney (MDCK) cells (Table 1 and Fig. 3). For some N-acylhydrazone compounds, we observed quite potent and selective activity in the vRNP reconstitution assay. This indicates that they are able to inhibit viral RNA synthesis and suggests that they could be classified as original PA inhibitors. Values for EC50 (vRNP) or EC90 (virus yield) in the range of 0.4–18 μM were obtained for compounds 15 and 20–23, which all carry a 3,4,5-trihydroxyphenyl as R2, and possess either two (15) or three (20–23) hydroxyl substituents in the R1 moiety. As in the enzymatic PA-Nter assays, the compounds having R2 as a gallic moiety (Fig. 3: 21, 22 and 23) showed slightly higher activity than the compounds carrying a 2-hydroxyl R2 group (9, 10 and 11); 10 and 22 have substantially the same EC50 in the vRNP reconstitution assay in HEK293T cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14990</offset><text>The hydrazide 28 displayed weak (virus yield) to moderate (vRNP reconstitution) activity, albeit less than the most active molecules in the 3,4,5-trihydroxyphenyl series (i.e. 18 and 21–23). Even if there are no data indicating that the compounds reported in the paper are subject to hydrolysis, the activity of 28 could raise the concern that for some N-acylhydrazones the antiviral activity in cell culture may be related to their intracellular hydrolysis. However, this is unlikely, since the antiviral potency showed large differences (i.e. EC50 values between 0.42 and 29 μM) for compounds with the same R2 but different R1 groups, meaning that R1 does play a role in modulating the antiviral effect.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15701</offset><text>Most compounds carrying as R1 a 2,3-dihydroxybenzylidene (i.e. 3, 5 and 7) or 2-hydroxy-3-methoxybenzylidene moiety (i.e. 4, 6 and 8) showed relatively high cytotoxicity in the vRNP assay, with CC50 values below 50 μM and a selectivity index (ratio of CC50 to EC50) below 8. Two notable exceptions are 18 and 19 (containing a 2,3-dihydroxybenzylidene or 2-hydroxy-3-methoxybenzylidene R1, respectively) which were not cytotoxic at 200 μM and displayed favorable antiviral selectivity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16192</offset><text>Some N-acylhydrazone compounds were devoid of activity in the enzymatic assay, yet showed good to moderate efficacy in cell culture (e.g. 14 and 19, having EC50 values of 2.2 and 7.1 μM, respectively). For most of the active compounds (i.e. 9, 11, 13, 15–21, 23, 24 and 26) a fair correlation was seen for the two cell-based assays, since the EC50 values obtained in the vRNP assay were maximum 5-fold different from the EC90 values in the virus yield assay. On the other hand, this difference was 8-fold or more for 7, 10, 14, 22, 25 and 28. Some N-acylhydrazone compounds showed good to moderate efficacy in the vRNP assay (e.g. 14 and 19, having EC50 values of 2.3 and 5.7 μM, respectively), yet were devoid of activity in the enzymatic assay. This observation suggests that they may inhibit the viral polymerase in an endonuclease-independent manner. To achieve a clear insight into the antiviral profile of the N-acylhydrazones, specific mechanistic experiments are currently ongoing in our laboratory, in which we are analyzing in full depth their effects on virus entry, polymerase-dependent RNA synthesis or the late stage (maturation and release) of the virus replication cycle.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>17388</offset><text>Docking studies</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17404</offset><text>In order to explore the possible binding mode of the synthesized compounds, docking simulations by GOLD program were performed by using the structural coordinates (PDB code 4AWM) for the PA-Nter endonuclease in complex with EGCG. Considering that the position of the side-chains of some residues changes depending on which pocket the ligand is occupying, we superimposed some X-ray structures of complexes between PA-Nter endonuclease and known active ligands. It was observed that the side-chain of amino acid Tyr24 shows greater movement than the other residues and for this reason we considered it as a flexible residue during the docking procedure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18057</offset><text>First, test docking calculations, using EGCG, L-742,001 and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one (Fig. 1), were carried out to compare experimental and predicted binding modes and validate docking procedure. Their best docking poses agreed well with the experimental binding modes (rmsd values of 0.8, 1.2 and 0.7, respectively).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18410</offset><text>Next, docking of several N-acylhydrazones was performed and this generated a number of possible binding conformations, highlighting that the active site cavity of the PA endonuclease is quite spacious, as already demonstrated by crystallographic studies, and confirming the ability of this scaffold to chelate the two M2+ ions in different ways (Mode A-C in Fig. 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18777</offset><text>Figure 5 displays the first (panel A) and second (panel B) GOLD cluster docked solutions for compound 23. These two complex structures represent the largest clusters with similar fitness values (59.20 and 58.65, respectively). In both cases, 23 appears able to coordinate the two M2+ ions in the active site through the three contiguous OH groups (Fig. 5). In addition, 23 was predicted to form two hydrogen bonding interactions, i.e. with the catalytic Lys134 on the one side and Glu26 on the other side. Furthermore, in these two different binding modes, 23 forms π–π interactions with the aromatic ring of Tyr24, in a fashion similar to that described for other endonuclease inhibitors, i.e. EGCG and L-742,001.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19496</offset><text>The best docked conformation for compound 15 (Fig. 6, fitness value 68.56), which has an activity slightly lower than 23, reveals a different role for the gallic moiety. The ligand seems to form two hydrogen bonding interactions with Tyr130 as well as a cation–π interaction with Lys134. Tyr130 lies in a pocket that also contains Arg124, a residue that was proposed to have a crucial role in binding of the RNA substrate. Compound 15 appears further stabilized by hydrogen bonding interactions between two hydroxyl groups and Arg82 and Asp108. In this case, chelation of the two M2+ ions is carried out by involving the imine group (mode A in Fig. 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20152</offset><text>It is important to highlight that compounds 23 and 15, although in different ways, both are able to chelate the metal cofactors and to establish interactions with highly conserved aminoacids (Tyr24, Glu26, Arg124, Tyr130 and Lys134) that are very important for both endonuclease activity and transcription in vitro. The crucial role of such interactions is underlined by the differences in activity between 15 (IC50 = 9.0 μM) and 19 (>500 μM): their coordinating features are similar, since both coordinate to the divalent metal ion through the phenolic oxygen, the iminic nitrogen and the carbonylic oxygen (mode A in Fig. 4), but the biological activity could be related to their different ability to engage interactions with the protein environment.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20915</offset><text>Crystallographic Studies</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20940</offset><text>Attempts were made to co-crystallize PA-Nter with 15, 20, 21 and 23 in one to four molar excess. While crystals appeared and diffracted well, upon data processing, no or very little electron density for the inhibitors was observed. Attempts to soak apo crystals in crystallization solution containing 5 mM inhibitor overnight also did not result in substantial electron density for the inhibitor. As a last resort, dry powder of the inhibitor was sprinkled over the crystallization drop containing apo crystals and left over night. This experiment was successful for compound 23, the crystals diffracted to 2.15 Å and diffraction data were collected (PDB ID 5EGA). The refined structure shows unambiguous electron density for the inhibitor (Table S1 and Fig. 7). The complex structure confirms one of the two binding modes predicted by the docking simulations (Fig. 5, panel B). The galloyl moiety chelates the manganese ions, while the trihydroxyphenyl group stacks against the Tyr24 side chain. It is interesting to note that two of these hydroxyl groups are in position to form hydrogen bonds with the side chain of Glu26 and Lys34 (Fig. 7). These interactions suggest that other functional groups, e.g. halogens, could be used in place of the hydroxyl groups for better interactions with Glu26 and Lys34 side chains, and the inhibitory potency of these compounds could be further improved.</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">title_1</infon><offset>22335</offset><text>Conclusions</text></passage><passage><infon key="section_type">CONCL</infon><infon key="type">paragraph</infon><offset>22347</offset><text>The development of new agents for the treatment of influenza infection that exert their action by inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is a strategy that recently is gaining a lot of interest. The results here presented add the N-acylhydrazone scaffold to the library of the chelating molecules with potent antiviral activity (EC90 < 5 μM, virus yield assay in influenza virus-infected MDCK cells). The structure of the N-acylhydrazone 23 co-crystallized with PA-Nter is important not only because confirms that the polyhydroxypheyl group efficiently coordinates two metal ions in the active site of the enzyme, but also because highlights the importance of the (flexible) inhibitor backbone in order to engage effective interactions with crucial aminoacids of the protein. Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase could represent another example, after carbonic anhydrase, histone deacetylase, and HIV-1 integrase, of metal binding as a successful strategy in drug design.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>23412</offset><text>Experimental Section</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>23433</offset><text>Materials and methods. Chemistry</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>23466</offset><text>All reagents of commercial quality were purchased from Sigma-Aldrich and used without further purification. The purity of the compounds was determined by elemental analysis and verified to be ≥95% for all synthesized molecules. NMR spectra were recorded at 25 °C on a Bruker Avance 400 FT spectrophotometer. The attenuate total reflectance IR spectra were recorded by means of a Nicolet-Nexus (Thermo Fisher) spectrophotometer by using a diamond crystal plate in the range of 4000–400 cm−1. Elemental analyses were performed by using a FlashEA 1112 series CHNS/O analyzer (Thermo Fisher) with gas-chromatographic separation. Electrospray mass spectral analyses (ESI-MS) were performed with an electrospray ionization (ESI) time-of-flight Micromass 4LCZ spectrometer. MS spectra were acquired in positive EI mode by means of a direct exposure probe mounting on the tip of a Re-filament with a DSQII Thermo Fisher apparatus, equipped with a single quadrupole analyzer. UV–Vis spectra were recorded on an Evolution 260 Bio Thermo spectrophotometer by using cells of 1 cm path length. UV-vis absorption spectra of 19 and 23 were registered using a ca. 10−5 M solution in methanol. Each metal/ligand system was studied by titrating a 2.8 ml sample of the ligand solution with a methanolic solution of Mg(CH3COO)2; 8–12 spectra of samples with M:L molar ratio ranging from 0 to 6 were measured.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24870</offset><text>Synthesis of the ligands (general procedure)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24915</offset><text>All the N-acylhydrazones were prepared in a manner similar to reported procedures. Briefly, to a solution of the aldehyde in absolute ethanol or toluene, an equimolar amount of the hydrazide dissolved in the same solvent was added. The mixture was refluxed for 6 hours, cooled at room temperature and concentrated in vacuum. The resulting precipitate was filtered off, washed with cold ethanol and dried in vacuum.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25330</offset><text>3,4,5-trihydroxybenzohydrazide (28) and 3,4,5-trimethoxybenzohydrazide (29) were obtained by reaction of the corresponding methyl esters with hydrazine monohydrate. Hydrazine was added to an ethanol suspension of the ester and stirred at room temperature until the solute completely dissolved. Reaction mixture was then refluxed overnight. On concentrating the solution, a precipitate was observed, which was filtered and washed with cold ethanol. Chemical characterization of 1–29 and of Mg(HL)2 4H2O is collected in the Supplementary Information.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>25881</offset><text>Computational Studies</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>25903</offset><text>The crystal structure of PA-Nter endonuclease in complex with EGCG was retrieved from the RCSB Protein Data Bank (entry code 4AWM). The ligand and water molecules were discarded and the hydrogens were added to the protein by Discovery Studio 2.5. The charge on the metal ions was set as +2. EGCG, L-742,001, and 2-(4-(1H-tetrazol-5-yl)phenyl)-5-hydroxypyrimidin-4(3H)-one structures were extracted from their X-ray complexes (PDB IDs 4AWM, 4W9S and 4E5H respectively). The other ligand structures were constructed using Discovery Studio 2.5.5 (Accelrys, Discovery Studio) and energy minimized using the Smart Minimizer protocol (1000 steps) which combines the Steepest Descent and the Conjugate Gradient methods.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>26618</offset><text>The minimized ligands were docked in their corresponding proteins by means of GOLD Suite 5.0.1. The region of interest used by the GOLD program was defined in order to contain the residues within 15 Å from the original position of the ligand in the X-ray structure. The side-chain of residue Tyr24 was allowed to rotate according to the internal rotamer libraries in GOLD Suite 5.0.1. GoldScore was chosen as fitness function. The standard default settings were used in all calculations and the ligands were submitted to 100 genetic algorithm runs. The “allow early termination” command was deactivated. Results differing by less than 0.75 Å in ligand-all atom rmsd, were clustered together. The best GOLD-calculated conformation was used both for analysis and representation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>27401</offset><text>Plasmid-based endonuclease assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>27434</offset><text>This enzymatic assay was performed according to a previously published method. One microgram of recombinant PA-Nter (residues 1–217 from the PA protein of influenza virus strain A/X-31) was incubated with 1 μg (16.7 nM) of single-stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, Louisiana) in the presence of the test compounds and at a final volume of 25 μL. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM β-mercaptoethanol and 1 mM MnCl2. The reaction was incubated at 37 °C for 2 h and then stopped by heat inactivation (80 °C, 20 min), followed by visualization of the endonucleolytic digestion of the plasmid by gel electrophoresis on a 1% agarose gel with ethidium bromide staining. The amount of remaining intact plasmid was quantified by ImageQuant TL software (GE Healthcare, Diegem, Belgium). The percentage inhibition of PA endonuclease activity was plotted against the compound concentration on a semi-logarithmic plot, using GraphPad Prism software (GraphPad Software, La Jolla, CA). The 50% inhibitory concentrations (IC50) were obtained by nonlinear least-squares regression analysis of the results from three independent experiments. 2,4-Dioxo-4-phenylbutanoic acid (DPBA; Interchim, Montluçon, France) was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>28740</offset><text>Cells and media</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>28756</offset><text>MDCK cells (a kind gift from M. Matrosovich, Marburg, Germany) and HEK293T cells (purchased from Thermo Fisher Scientific, Waltham, MA) were cultivated in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and 0.075% sodium bicarbonate. During virus experiments, the MDCK cells were maintained in MDCK infection medium, consisting of Ultra MDCK medium (Lonza, Basel, Switzerland) supplemented with 0.0225% sodium bicarbonate, 2 mM L-glutamine, and 2 μg/ml tosyl phenylalanyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO). The cells were incubated in a humidified atmosphere containing 5% CO2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>29417</offset><text>vRNP reconstitution assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>29443</offset><text>The procedure to determine the inhibitory effect of the compounds on influenza virus vRNPs reconstituted in HEK293T cells, is described in full detail elsewhere. Briefly, the four relevant plasmids (i.e. the expression plasmids for PB1, PB2, PA and NP) were combined with the fluc reporter plasmid, and co-transfected into HEK293T cells using Lipofectamin 2000 (Invitrogen, Life Technologies, Gent, Belgium). After incubation at 37 °C for 24 h in the presence of serial dilutions of the test compounds, the ONE-Glo luciferase assay system (Promega, Madison, WI) was used to determine luciferase activity. EC50 was defined as the compound concentration causing 50% reduction in the vRNP-driven firefly luciferase signal, as compared to cells receiving medium instead of compound. These EC50 values were calculated by interpolation assuming a semi-log dose-response effect using GraphPad Prism software. In parallel, compound cytotoxic activity was determined in untransfected HEK293T cells which had been incubated with serial dilutions of the compounds for 24 h, using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega). These spectrophotometric data were used to calculate the 50% cytotoxic concentration (CC50), i.e. the concentration reducing cell viability by 50%, as compared to wells receiving medium instead of compound. Ribavirin (Virazole; ICN Pharmaceuticals, Costa Mesa, CA) was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31011</offset><text>Virus yield assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31029</offset><text>We previously published in full detail the virus yield assay to determine the anti-influenza virus activity in MDCK cell cultures. Briefly, one day prior to infection, MDCK cells were seeded into 96-well plates at 25,000 cells per well. At day 0, serial dilutions of the test compounds were added, immediately followed by infection with influenza A/PR/8/34 virus. After 24 h incubation at 35 °C, the virus amount in the supernatants was estimated by determining the viral genome copy number in a one-step quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assay (CellsDirect One-Step qRT-PCR kit; Invitrogen), with influenza virus M1-specific primers and probe. The compound concentration values causing a 2-log10 (EC99) and a 1-log10 (EC90) reduction in viral RNA (vRNA) copy number at 24 h p.i., as compared to the virus control receiving no compound, were calculated by interpolation from data of at least three experiments. In parallel, the CC50 values after 24 h incubation with compounds were determined in uninfected MDCK cells, using the spectrophotometric MTS cell viability assay described above, respectively. Ribavirin was included as the reference compound.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32233</offset><text>Crystallographic analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32259</offset><text>A PAN construct (PANΔLoop) with a loop (residues 51–72) deleted and replaced with GGS from A/California/04/2009 H1N1 strain was used for the crystallographic studies. The details of cloning, over-expression and purification are described elsewhere. Briefly, the gene was cloned into pET52b vector and transformed into BL21 (DE3) cells, and the protein was expressed in LB medium overnight at 18 °C after induction at an OD600 ~0.8 with 0.2 mM isopropyl-β-thiogalactopyranoside (IPTG). The protein was purified from cell lysates by HisTrap affinity chromatography and the 10xHis tag was removed by digestion with thrombin. The protein was further purified by gel filtration using a Superdex 75 size-exclusion chromatography column in 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM TCEP. The protein was concentrated to 10–12 mg/ml for crystallization. Crystals were grown in 0.2 M MgCl2, 2 mM MnCl2, 0.1 M Tris pH 8.5, 30% (w/v) PEG 4000 using the hanging drop method. For determination of the protein-inhibitor complex structure, the powder of the inhibitor was sprinkled on a 2 μl drop of a 1:1 ratio mixture of protein solution and well solution, on a cover slide hanging over 500 μl well solution, and left overnight. Next day, the crystals were cryo-protected using well solution supplemented with 25% ethylene glycol and flash frozen in liquid nitrogen. The data were collected at the 22-ID beam line maintained by Southeast Regional Collaborative Access Team (SERCAT) at the Advanced Photon Source, Argonne National Laboratory. The data were indexed, integrated and scaled using the HKL2000 suite of programs. Phase determination, structure refinement and model building were completed using Phaser, Refmac and Coot (part of the CCP4 package). The apo structure of PANΔLoop (PDB ID: 5DES) was used as starting model for molecular replacement. The details of the data collection and refinement statistics are given in Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34198</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34221</offset><text>How to cite this article: Carcelli, M. et al. N-acylhydrazone inhibitors of influenza virus PA endonuclease with versatile metal binding modes. Sci. 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All authors have given approval to the final version of the manuscript. M.C., D.R., A.G. and M.S. drug design and chemical synthesis; L.D.L. docking studies; G.K. and S.W.W. crystallographic studies; A.S. and L.N. biological studies.</text></passage><passage><infon key="file">srep31500-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40191</offset><text>Chemical structures of some prototype inhibitors of influenza virus endonuclease.</text></passage><passage><infon key="file">srep31500-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40273</offset><text>Inhibitor activity in enzymatic assays (IC50, μM) as reported in: aref., bref., cref., dref..</text></passage><passage><infon key="file">srep31500-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40370</offset><text>General synthesis for N-acylhydrazones 1–27 and hydrazides 28 and 29 (A). Chemical structures of compounds 1–27 (B).</text></passage><passage><infon key="file">srep31500-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40491</offset><text>Overview of the structure-activity relationship for compounds 1–27.</text></passage><passage><infon key="file">srep31500-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>40561</offset><text>Scheme of possible binding modes of the studied N-acylhydrazones.</text></passage><passage><infon key="file">srep31500-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>40627</offset><text>First (A) and second (B) GOLD cluster docked solutions of compound 23 (orange and cyan, respectively) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
2011,
51, 2778]. Hydrogen bonds are illustrated by dotted lines, while the divalent metal ions are shown as purple spheres. Schematic drawings of the interactions of the first (C) and second (D) GOLD cluster docked solutions generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text></passage><passage><infon key="file">srep31500-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41286</offset><text>(A) Binding mode of compound 15 (orange) in complex with PA endonuclease. Key residues of the pocket are presented using PyMOL [ http://www.pymol.org] and LIGPLUS [Laskowski, R. A.; Swindells, M. B. Journal of chemical information and modeling
2011,
51, 2778]. Hydrogen bonds are illustrated by dotted lines while the divalent metal ions are shown as purple spheres. (B) Schematic drawing of the interactions of compound 15 generated using LIGPLUS. Dashed lines are hydrogen bonds and ‘eyelashes’ show residues involved in hydrophobic interactions.</text></passage><passage><infon key="file">srep31500-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>41839</offset><text>Crystal structure of PANΔLoop in complex with compound 23.</text></passage><passage><infon key="file">srep31500-f7.jpg</infon><infon key="id">f7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>41902</offset><text>Active site residues are shown in sticks with green carbons, manganese atoms are shown as purple spheres and water molecules as red spheres. Compound 23 is shown in sticks with yellow carbons. Protein secondary structure is shown as ribbons in salmon color. 2Fo-Fc electron density map contoured at 1σ is shown as blue mesh. Hydrogen bonds and metal coordination are shown with dotted lines. The H-bond distances from the side chain carboxyl group of Glu26 to p-OH and m-OH of the trihydroxyphenyl group of the inhibitor are 2.7 Å and 3.0 Å, respectively. The H-bond distance from the side chain of Lys34 to p-OH of the trihydroxyphenyl group is 3.6 Å. The H-bond distance to the water molecule from m-OH of the galloyl moiety is 3.0 Å, which in turn is H-bonded to the side chain of Tyr130 with a distance of 2.7 Å. Crystal structure has been deposited in the RCSB Protein Data Bank with PDB ID: 5EGA.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>42811</offset><text>Inhibitory activity of the N-acylhydrazones 1–27 and hydrazide 28 in the enzymatic assay with influenza virus PA-Nter endonuclease, or in cellular influenza virus assays.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th rowspan="3" align="left" valign="top" charoff="50">Compound</th><th rowspan="2" align="center" valign="top" charoff="50">Enzyme assay with PA-Nter<xref ref-type="fn" rid="t1-fn1">a</xref></th><th colspan="4" align="center" valign="top" charoff="50">Virus yield assay in influenza virus-infected MDCK cells<xref ref-type="fn" rid="t1-fn2">b</xref></th><th colspan="2" align="center" valign="top" charoff="50">vRNP reconstitution assay in HEK293T cells<xref ref-type="fn" rid="t1-fn3">c</xref></th></tr><tr><th colspan="2" align="center" valign="top" charoff="50">Antiviral activity</th><th align="center" valign="top" charoff="50">Cytotoxicity</th><th rowspan="2" align="center" valign="top" charoff="50">SI<xref ref-type="fn" rid="t1-fn4">d</xref></th><th align="center" valign="top" charoff="50">Activity</th><th align="center" valign="top" charoff="50">Cytotoxicity</th></tr><tr><th align="center" valign="top" charoff="50">IC<sub>50</sub></th><th align="center" valign="top" charoff="50">EC<sub>99</sub></th><th align="center" valign="top" charoff="50">EC<sub>90</sub></th><th align="center" valign="top" charoff="50">CC<sub>50</sub></th><th align="center" valign="top" charoff="50">EC<sub>50</sub></th><th align="center" valign="top" charoff="50">CC<sub>50</sub></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><bold>(1)</bold></td><td align="center" valign="top" charoff="50">24</td><td align="center" valign="top" charoff="50">ND<xref ref-type="fn" rid="t1-fn6">f</xref></td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">107</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(2)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">&gt;100</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(3)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">5.9</td><td align="center" valign="top" charoff="50">48</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(4)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">6.3</td><td align="center" valign="top" charoff="50">33</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(5)</bold></td><td align="center" valign="top" charoff="50">67</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">≥146</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">2.6</td><td align="center" valign="top" charoff="50">10</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(6)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;50</td><td align="center" valign="top" charoff="50">&gt;50</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">15</td><td align="center" valign="top" charoff="50">14</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(7)</bold></td><td align="center" valign="top" charoff="50">54</td><td align="center" valign="top" charoff="50">172</td><td align="center" valign="top" charoff="50">100</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.0</td><td align="center" valign="top" charoff="50">3.2</td><td align="center" valign="top" charoff="50">8.9</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(8)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">1.9</td><td align="center" valign="top" charoff="50">15</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(9)</bold></td><td align="center" valign="top" charoff="50">34</td><td align="center" valign="top" charoff="50">16</td><td align="center" valign="top" charoff="50">5.3</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;38</td><td align="center" valign="top" charoff="50">5.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(10)</bold></td><td align="center" valign="top" charoff="50">68</td><td align="center" valign="top" charoff="50">14</td><td align="center" valign="top" charoff="50">8.5</td><td align="center" valign="top" charoff="50">111</td><td align="center" valign="top" charoff="50">&gt;13</td><td align="center" valign="top" charoff="50">0.40</td><td align="center" valign="top" charoff="50">132</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(11)</bold></td><td align="center" valign="top" charoff="50">45</td><td align="center" valign="top" charoff="50">30</td><td align="center" valign="top" charoff="50">12</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;17</td><td align="center" valign="top" charoff="50">5.6</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(12)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;12.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">20</td><td align="center" valign="top" charoff="50">39</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(13)</bold></td><td align="center" valign="top" charoff="50">69</td><td align="center" valign="top" charoff="50">71</td><td align="center" valign="top" charoff="50">34</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;5.9</td><td align="center" valign="top" charoff="50">6.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(14)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">63</td><td align="center" valign="top" charoff="50">37</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;5.4</td><td align="center" valign="top" charoff="50">2.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(15)</bold></td><td align="center" valign="top" charoff="50">8.9</td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">7.5</td><td align="center" valign="top" charoff="50">≥172</td><td align="center" valign="top" charoff="50">≥23</td><td align="center" valign="top" charoff="50">14</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(16)</bold></td><td align="center" valign="top" charoff="50">454</td><td align="center" valign="top" charoff="50">67</td><td align="center" valign="top" charoff="50">28</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.1</td><td align="center" valign="top" charoff="50">5.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(17)</bold></td><td align="center" valign="top" charoff="50">482</td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">8.1</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;25</td><td align="center" valign="top" charoff="50">7.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(18)</bold></td><td align="center" valign="top" charoff="50">83</td><td align="center" valign="top" charoff="50">6.2</td><td align="center" valign="top" charoff="50">2.2</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;91</td><td align="center" valign="top" charoff="50">3.3</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(19)</bold></td><td align="center" valign="top" charoff="50">&gt;500</td><td align="center" valign="top" charoff="50">53</td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.7</td><td align="center" valign="top" charoff="50">5.7</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(20)</bold></td><td align="center" valign="top" charoff="50">18</td><td align="center" valign="top" charoff="50">35</td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;18</td><td align="center" valign="top" charoff="50">2.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(21)</bold></td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">8.3</td><td align="center" valign="top" charoff="50">3.6</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;56</td><td align="center" valign="top" charoff="50">2.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(22)</bold></td><td align="center" valign="top" charoff="50">75</td><td align="center" valign="top" charoff="50">7.4</td><td align="center" valign="top" charoff="50">3.4</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;59</td><td align="center" valign="top" charoff="50">0.42</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(23)</bold></td><td align="center" valign="top" charoff="50">8.7</td><td align="center" valign="top" charoff="50">11</td><td align="center" valign="top" charoff="50">3.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;57</td><td align="center" valign="top" charoff="50">3.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(24)</bold></td><td align="center" valign="top" charoff="50">131</td><td align="center" valign="top" charoff="50">58</td><td align="center" valign="top" charoff="50">26</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;7.7</td><td align="center" valign="top" charoff="50">25</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(25)</bold></td><td align="center" valign="top" charoff="50">40</td><td align="center" valign="top" charoff="50">132</td><td align="center" valign="top" charoff="50">70</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.9</td><td align="center" valign="top" charoff="50">4.1</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(26)</bold></td><td align="center" valign="top" charoff="50">30</td><td align="center" valign="top" charoff="50">36</td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;15</td><td align="center" valign="top" charoff="50">5.5</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(27)</bold></td><td align="center" valign="top" charoff="50">36</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">21</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50"><bold>(28)</bold></td><td align="center" valign="top" charoff="50">40</td><td align="center" valign="top" charoff="50">158</td><td align="center" valign="top" charoff="50">85</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;2.4</td><td align="center" valign="top" charoff="50">7.2</td><td align="center" valign="top" charoff="50">&gt;200</td></tr><tr><td align="left" valign="top" charoff="50">DPBA<xref ref-type="fn" rid="t1-fn5">e</xref></td><td align="center" valign="top" charoff="50">5.3</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">ND</td></tr><tr><td align="left" valign="top" charoff="50">Ribavirin</td><td align="center" valign="top" charoff="50">ND</td><td align="center" valign="top" charoff="50">13</td><td align="center" valign="top" charoff="50">8.5</td><td align="center" valign="top" charoff="50">&gt;200</td><td align="center" valign="top" charoff="50">&gt;24</td><td align="center" valign="top" charoff="50">9.4</td><td align="center" valign="top" charoff="50">&gt;200</td></tr></tbody></table>
</infon><offset>42984</offset><text>Compound Enzyme assay with PA-Ntera Virus yield assay in influenza virus-infected MDCK cellsb vRNP reconstitution assay in HEK293T cellsc Antiviral activity Cytotoxicity SId Activity Cytotoxicity IC50 EC99 EC90 CC50 EC50 CC50 (1) 24 NDf ND ND 107 >200 (2) >500 ND ND ND >100 >200 (3) >500 ND ND >200 5.9 48 (4) >500 ND ND >200 6.3 33 (5) 67 >25 >25 ≥146 2.6 10 (6) >500 >50 >50 >200 15 14 (7) 54 172 100 >200 >2.0 3.2 8.9 (8) >500 >12.5 >12.5 >200 1.9 15 (9) 34 16 5.3 >200 >38 5.5 >200 (10) 68 14 8.5 111 >13 0.40 132 (11) 45 30 12 >200 >17 5.6 >200 (12) >500 >12.5 >12.5 >200 20 39 (13) 69 71 34 >200 >5.9 6.3 >200 (14) >500 63 37 >200 >5.4 2.3 >200 (15) 8.9 18 7.5 ≥172 ≥23 14 >200 (16) 454 67 28 >200 >7.1 5.2 >200 (17) 482 21 8.1 >200 >25 7.1 >200 (18) 83 6.2 2.2 >200 >91 3.3 >200 (19) >500 53 26 >200 >7.7 5.7 >200 (20) 18 35 11 >200 >18 2.2 >200 (21) 13 8.3 3.6 >200 >56 2.5 >200 (22) 75 7.4 3.4 >200 >59 0.42 >200 (23) 8.7 11 3.5 >200 >57 3.1 >200 (24) 131 58 26 >200 >7.7 25 >200 (25) 40 132 70 >200 >2.9 4.1 >200 (26) 30 36 13 >200 >15 5.5 >200 (27) 36 ND ND ND 21 >200 (28) 40 158 85 >200 >2.4 7.2 >200 DPBAe 5.3 ND ND ND ND ND Ribavirin ND 13 8.5 >200 >24 9.4 >200 </text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>44265</offset><text>aRecombinant PA-Nter was incubated with the ssDNA plasmid substrate, a Mn2+-containing buffer and test compounds. Cleavage of the substrate was assessed after 2 hr incubation. The IC50 represents the compound concentration (in μM) required to obtain 50% inhibition of cleavage, calculated by nonlinear least-squares regression analysis (using GraphPad Prism software) of the results from 2–4 independent experiments.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>44687</offset><text>bMDCK cells were infected with influenza A virus (strain A/PR/8/34) and incubated with the compounds during 24 h. The virus yield in the supernatant was assessed by real-time qPCR. The EC99 and EC90 values represent the compound concentrations (in μM) producing a 2-log10 or 1-log10 reduction in virus titer, respectively, determined in 2–3 independent experiments. The cytotoxicity, assessed in uninfected MDCK cells, was expressed as the CC50 value (50% cytotoxic concentration, determined with the MTS cell viability assay, in μM).</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45226</offset><text>cHEK293T cells were co-transfected with the four vRNP-reconstituting plasmids and the luciferase reporter plasmid in the presence of the test compounds. The EC50 represents the compound concentration (in μM) producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 h after transfection. The EC50 value was derived from data from 2–4 independent experiments, by nonlinear least-squares regression analysis (using GraphPad Prism software). The CC50 (in μM), i.e. the 50% cytotoxic concentration, was determined in untransfected HEK293T cells by MTS cell viability assay.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45824</offset><text>dSI, selectivity index, defined as the ratio between the CC50 and EC90.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45896</offset><text>eDPBA, 2,4-dioxo-4-phenylbutanoic acid.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>45936</offset><text>fND, not determined.</text></passage></document></collection>
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<collection><source>PMC</source><date>20201217</date><key>pmc.key</key><document><id>4981400</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</infon><infon key="article-id_doi">10.1371/journal.pone.0160694</infon><infon key="article-id_pmc">4981400</infon><infon key="article-id_pmid">27513867</infon><infon key="article-id_publisher-id">PONE-D-16-20928</infon><infon key="elocation-id">e0160694</infon><infon key="issue">8</infon><infon key="license">This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="name_0">surname:Zhang;given-names:Yinglu</infon><infon key="name_1">surname:Rataj;given-names:Katarzyna</infon><infon key="name_2">surname:Simpson;given-names:Gordon G.</infon><infon key="name_3">surname:Tong;given-names:Liang</infon><infon key="name_4">surname:Candela;given-names:Hector</infon><infon key="name_5">surname:Tong;given-names:Liang</infon><infon key="name_6">surname:Tong;given-names:Liang</infon><infon key="name_7">surname:Simpson;given-names:Gordon G.</infon><infon key="name_8">surname:Simpson;given-names:Gordon G.</infon><infon key="notes">All relevant data are within the paper and its Supporting Information files.</infon><infon key="section_type">TITLE</infon><infon key="title">Data Availability</infon><infon key="type">front</infon><infon key="volume">11</infon><infon key="year">2016</infon><offset>0</offset><text>Crystal Structure of the SPOC Domain of the Arabidopsis Flowering Regulator FPA</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>80</offset><text>The Arabidopsis protein FPA controls flowering time by regulating the alternative 3′-end processing of the FLOWERING LOCUS (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. The overall structure is similar to that of the SPOC domain in human SMRT/HDAC1 Associated Repressor Protein (SHARP), although there are also substantial conformational differences between them. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation or the functions of the FPA SPOC domain cannot be disrupted by the combination of mutations, in contrast to observations with the SHARP SPOC domain.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1277</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1290</offset><text>Eukaryotic messenger RNAs (mRNAs) are made as precursors through transcription by RNA polymerase II (Pol II), and these primary transcripts undergo extensive processing, including 3′-end cleavage and polyadenylation. In addition, alternative 3′-end cleavage and polyadenylation is an essential and ubiquitous process in eukaryotes. Misregulation of (alternative) 3′-end processing can lead to various genetic defects, cancer and other diseases. There is currently great interest in understanding the molecular mechanisms and functional impacts of alternative 3′-end processing.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1876</offset><text>Recently, the split ends (SPEN) family of proteins was identified as RNA binding proteins that regulate alternative 3′-end cleavage and polyadenylation. They are characterized by possessing N-terminal RNA recognition motifs (RRMs) and a conserved SPEN paralog and ortholog C-terminal (SPOC) domain (Fig 1A). The SPOC domain is believed to mediate protein-protein interactions and has diverse functions among SPEN family proteins, but the molecular mechanism of these functions is not well understood.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>2379</offset><text>Sequence conservation of SPOC domains.</text></passage><passage><infon key="file">pone.0160694.g001.jpg</infon><infon key="id">pone.0160694.g001</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>2418</offset><text>(A). Domain organization of A. thaliana FPA. (B). Sequence alignment of the SPOC domains of Arabidopsis thaliana FPA, human RBM15, Drosophila SPEN, mouse MINT, and human SHARP. Residues in surface patch 1 are indicated with the orange dots, and those in surface patch 2 with the green dots. The secondary structure elements in the structure of FPA SPOC are labeled. Residues that are strictly conserved among the five proteins are shown in white with a red background, and those that are mostly conserved in red.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2931</offset><text>FPA, a SPEN family protein in Arabidopsis thaliana and other plants, was found to regulate the 3′-end alternative cleavage and polyadenylation of the antisense RNAs of FLOWERING LOCUS (FLC), a flowering repressor gene. FPA promotes the 3′-end processing of class I FLC antisense RNAs, which includes the proximal polyadenylation site. This is associated with histone demethylase activity and down-regulation of FLC transcription. However, the functional mechanism of this complex is still not clear.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3435</offset><text>Although a SPOC domain is found in all the SPEN family proteins, its sequence conservation is rather low. For example, the sequence identity between the SPOC domains of A. thaliana FPA and human SMRT/HDAC1 Associated Repressor Protein (SHARP) is only 19% (Fig 1B). Currently, the SHARP SPOC domain is the only one with structural information.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3778</offset><text>As a first step toward understanding the molecular basis for the regulation of alternative 3′-end processing and flowering by FPA, we have determined the crystal structure of the SPOC domain of A. thaliana FPA at 2.7 Å resolution. The overall structure is similar to that of the SHARP SPOC domain, although there are also substantial conformational differences between them. The structure reveals a surface patch that is conserved among FPA homologs.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4232</offset><text>Results and Discussion</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>4255</offset><text>Structure of FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4284</offset><text>The crystal structure of the SPOC domain of A. thaliana FPA has been determined at 2.7 Å resolution using the selenomethionyl single-wavelength anomalous dispersion method. The expression construct contained residues 433–565 of FPA, but only residues 439–460 and 465–565 are ordered in the crystal. The atomic model has good agreement with the X-ray diffraction data and the expected bond lengths, bond angles and other geometric parameters (Table 1). All the residues are located in the favored regions of the Ramachandran plot (data not shown). The structure has been deposited in the Protein Data Bank, with accession code 5KXF.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>4923</offset><text>Summary of crystallographic information.</text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><colgroup span="1"><col align="left" valign="middle" span="1"/><col align="left" valign="middle" span="1"/></colgroup><tbody><tr><td align="left" rowspan="1" colspan="1">Resolution range (Å)<xref ref-type="table-fn" rid="t001fn001"><sup>1</sup></xref></td><td align="center" rowspan="1" colspan="1">50–2.7 (2.8–2.7)</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of observations</td><td align="center" rowspan="1" colspan="1">78,008</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub> (%)</td><td align="center" rowspan="1" colspan="1">10.5 (45.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">I/σI</td><td align="center" rowspan="1" colspan="1">24.1 (6.3)</td></tr><tr><td align="left" rowspan="1" colspan="1">Redundancy</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">Completeness (%)</td><td align="center" rowspan="1" colspan="1">100 (100)</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>R</italic> factor (%)</td><td align="center" rowspan="1" colspan="1">19.2 (25.0)</td></tr><tr><td align="left" rowspan="1" colspan="1">Free <italic>R</italic> factor (%)</td><td align="center" rowspan="1" colspan="1">25.4 (35.4)</td></tr><tr><td align="left" rowspan="1" colspan="1">Rms deviation in bond lengths (Å)</td><td align="center" rowspan="1" colspan="1">0.017</td></tr><tr><td align="left" rowspan="1" colspan="1">Rms deviation in bond angles (°)</td><td align="center" rowspan="1" colspan="1">1.9</td></tr></tbody></table>
</infon><offset>4964</offset><text>Resolution range (Å)1 50–2.7 (2.8–2.7) Number of observations 78,008 Rmerge (%) 10.5 (45.3) I/σI 24.1 (6.3) Redundancy Completeness (%) 100 (100) R factor (%) 19.2 (25.0) Free R factor (%) 25.4 (35.4) Rms deviation in bond lengths (Å) 0.017 Rms deviation in bond angles (°) 1.9 </text></passage><passage><infon key="file">pone.0160694.t001.xml</infon><infon key="id">pone.0160694.t001</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>5272</offset><text>1The numbers in parentheses are for the highest resolution shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5338</offset><text>The crystal structure of the FPA SPOC domain contains a seven-stranded, mostly anti-parallel β-barrel (β1-β7) and three helices (αA-αC) (Fig 2A). Only two of the neighboring strands, β1 and β3, are parallel to each other. Helix αB covers one end of the barrel, while helices αA and αC are located next to each other at one side of the barrel (Fig 2B). The other end of the β-barrel is covered by the loop connecting strands β2 and β3, which contains the disordered 461–464 segment. The center of the barrel is filled with hydrophobic side chains and is not accessible to the solvent.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>5966</offset><text>Crystal structure of the SPOC domain of A. thaliana FPA.</text></passage><passage><infon key="file">pone.0160694.g002.jpg</infon><infon key="id">pone.0160694.g002</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6023</offset><text>(A). Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (B). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (www.pymol.org).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>6471</offset><text>Comparisons to structural homologs of the SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>6525</offset><text>Only five structural homologs of the FPA SPOC domain were found in the Protein Data Bank with the DaliLite server, suggesting that the SPOC domain structure is relatively unique. The top hit is the SPOC domain of human SHARP (Fig 3A), with a Z score of 12.3. The other four structural homologs include the β-barrel domain of the proteins Ku70 and Ku80 (Z score 11.4) (Fig 3B), a domain in the chromodomain protein Chp1 (Z score 10.8) (Fig 3C), and the activator interacting domain (ACID) of the Med25 subunit of the Mediator complex (Z score 8.5) (Fig 3D). The next structural homolog has a Z score of 3.0.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>7135</offset><text>Structural homologs of the FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g003.jpg</infon><infon key="id">pone.0160694.g003</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>7179</offset><text>(A). Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. (B). Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. (C). Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (D). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8127</offset><text>SHARP is a transcriptional co-repressor in the nuclear receptor and Notch/RBP-Jκ signaling pathways. The SPOC domain of SHARP interacts directly with silencing mediator for retinoid and thyroid receptor (SMRT), nuclear receptor co-repressor (N-CoR), HDAC, and other components to represses transcription. While the overall structure of the FPA SPOC domain is similar to that of the SHARP SPOC domain, there are noticeable differences in the positioning of the β-strands and the helices, and most of the loops have substantially different conformations as well (Fig 3A). In addition, the SHARP SPOC domain has three extra helices. One of them covers the other end of the β-barrel, and the other two shield an additional surface of the side of the β-barrel from solvent. A doubly-phosphorylated peptide from SMRT is bound to the side of the barrel, near strands β1 and β3 (Fig 3A). Such a binding mode probably would not be possible in FPA, as the peptide would clash with the β1-β2 loop.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9139</offset><text>The Ku70-Ku80 hetero-dimer is involved in DNA double-strand break repair and the β-barrel domain contributes to DNA binding. In fact, the β-barrel domains of Ku70 and Ku80 form a hetero-dimer, primarily through interactions between the loops connecting the third and fourth strands of the barrel (Fig 3B). The open ends of the two β-barrels face the DNA binding sites, and contact the phosphodiester backbone of the dsDNA. In addition, a long insert connecting strands β2 and β3 in the two domains form an arch-like structure, encircling the dsDNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9703</offset><text>Chp1 is a subunit of the RNA-induced initiation of transcriptional gene silencing (RITS) complex. The partner of Chp1, Tas3, is bound between the barrel domain and the second domain of Chp1, and the linker between the two domains is also crucial for this interaction (Fig 3C). It is probably unlikely that the β-barrel itself is sufficient to bind Tas3. Interestingly, a loop in Tas3 contacts strand β3 of the barrel domain, at a location somewhat similar to that of the N-terminal segment of the SMRT peptide in complex with SHARP SPOC domain (Fig 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10263</offset><text>Mediator is a coactivator complex that promotes transcription by Pol II. The Med25 subunit ACID is the target of the potent activator VP16 of the herpes simplex virus. The structure of ACID contains a helix at the C-terminus as well as an extended β1-β2 loop. Nonetheless, the binding site for VP16 has been mapped to roughly the same surface patch, near strands β1 and β3, that is used by the SHARP and Tas3 SPOC domains for binding their partners.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10725</offset><text>A conserved surface patch in the FPA SPOC domain</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10774</offset><text>An analysis of the SPOC domain indicates a large surface patch near strands β1, β3, β5 and β6 that is conserved among plant FPA homologs (Fig 4A). This surface patch can be broken into two sub-patches, with residues Lys447 (in strand β1), Arg477 (β3), Tyr515 (αB) and Arg521 (β5) in one sub-patch, and residues His486 (αA), Thr478 (β3), Val524 (β5) and Phe534 (β6) in the other sub-patch (Fig 4B). The first surface patch is electropositive in nature (Fig 4C), and residues Arg477 and Tyr515 are also conserved in the SHARP SPOC domain (Fig 1B). In fact, one of the phosphorylated residues of the SMRT peptide interacts with this surface patch (Fig 3A), suggesting that the FPA SPOC domain might also interact with a phosphorylated segment here. In comparison, the second surface patch is more hydrophobic in nature (Fig 4C).</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11638</offset><text>A conserved surface patch of FPA SPOC domain.</text></passage><passage><infon key="file">pone.0160694.g004.jpg</infon><infon key="id">pone.0160694.g004</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11684</offset><text>(A). Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. (B). Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. (C). Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12177</offset><text>Testing the requirement of specific conserved amino acids for FPA functions</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12253</offset><text>We next examined the potential impact of the conserved surface patch on FPA function in vivo. We mutated two residues, Arg477 and Tyr515, of the surface patch, which are also conserved in the SHARP SPOC domain (Fig 1B) and were found to be functionally important. The mutations were introduced into a transgene designed to express FPA from its native control elements (promoter, introns and 3′ UTR). The resulting transgenes were then stably transformed into an fpa-8 mutant background so that the impact of the mutations on FPA function could be assessed. Control transformation of the same expression constructs into fpa-8 designed to express wild-type FPA protein restored FPA protein expression levels to near wild-type levels (panel A in S1 Fig) and rescued the function of FPA in controlling RNA 3′-end formation, for example in FPA pre-mRNA (panel B in S1 Fig). We examined independent transgenic lines expressing each R477A and Y515A mutation. In each case, we confirmed that detectable levels of FPA protein expression were restored close to wild-type levels in protein blot analyses using antibodies that specifically recognize FPA (S2 Fig).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13409</offset><text>We then examined the impact of the surface patch mutations on FPA’s function in controlling RNA 3′-end formation by determining whether the mutant proteins functioned in FPA autoregulation and the repression of FLC expression. FPA autoregulates its expression by promoting cleavage and polyadenylation within intron 1 of its own pre-mRNA, resulting in a truncated transcript that does not encode functional protein. We used RNA gel blot analyses to reveal that in each of three independent transgenic lines for each single mutant, rescue of proximally polyadenylated FPA pre-mRNA can be detected (Fig 5A and 5B). We therefore conclude that neither of these mutations disrupted the ability of FPA to promote RNA 3′-end formation in its own transcript.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14166</offset><text>Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.</text></passage><passage><infon key="file">pone.0160694.g005.jpg</infon><infon key="id">pone.0160694.g005</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14261</offset><text>RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing either FPA::FPA R477A
(A), or FPA::FPA Y515A
(B) using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. Proximally and distally polyadenylated FPA transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. (C,D) Impact of individual FPA SPOC domain mutations on FLC transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, 35S::FPA:YFP and FPA::FPA R477A
(C), FPA::FPA Y515A
(D) plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15153</offset><text>We next examined whether the corresponding mutations disrupted the ability of FPA to control FLC expression. We used RT-qPCR to measure the expression of FLC mRNA and found that in each independent transgenic line encoding each mutated FPA protein, the elevated levels of FLC detected in fpa-8 mutants were restored to near wild-type levels by expression of the FPA SPOC conserved patch mutant proteins (Fig 5C and 5D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15573</offset><text>Since each surface patch mutation appeared to be insufficient to disrupt FPA functions on its own, we combined both mutations into the same transgene. We could again confirm that near wild-type levels of FPA protein were expressed from three independent transgenic lines expressing the FPA R477A;Y515A doubly mutated protein in an fpa-8 mutant background (S3 Fig). We found that FPA R477A;Y515A protein functioned like wild-type FPA to restore FPA pre-mRNA proximal polyadenylation (Fig 6A) and FLC expression to wild-type levels (Fig 6B).</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16113</offset><text>Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and FLC expression.</text></passage><passage><infon key="file">pone.0160694.g006.jpg</infon><infon key="id">pone.0160694.g006</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16223</offset><text>(A) RNA gel blot analysis of WT A. thaliana accession Columbia (Col-0) plants fpa-8 and fpa-8 mutants expressing FPA::FPA R477A;Y515A using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated FPA mRNAs. A probe corresponding to the 5’UTR region of FPA mRNA was used to detect FPA specific mRNAs. RNA size (kb) marker (Ambion). TUBULIN was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. (B). qRT-PCR analysis was performed with total RNA purified from Col-0, fpa-8, and FPA::FPA R477A;Y515A plants. Transcript levels were normalized to the control UBC. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16983</offset><text>Together our findings suggest that either the SPOC domain is not required for the role of FPA in regulating RNA 3′-end formation, or that this combination of mutations is not sufficient to critically disrupt the function of the FPA SPOC domain. Since the corresponding mutations in the SHARP SPOC domain do disrupt its recognition of unphosphorylated SMRT peptides, these observations may reinforce the idea that the features and functions of the FPA SPOC domain differ from those of the only other well-characterized SPOC domain.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>17516</offset><text>Materials and Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>17538</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17574</offset><text>The SPOC domain (residue 433–565) of A. thaliana FPA was sub-cloned into the pET28a vector (Novagen). The recombinant protein, with an N-terminal hexa-histidine tag, was over-expressed in E. coli BL21 Star (DE3) cells (Novagen), which were induced with 0.4 mM IPTG and allowed to grow at 20°C for 14–18 h. The soluble protein was purified by nickel-charged immobilized-metal affinity chromatography and gel filtration chromatography. The purified protein was concentrated and stored at –80°C in a buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 10 mM DTT and 5% (v/v) glycerol. The His-tag was not removed for crystallization.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18213</offset><text>The selenomethionine labeled SPOC domain was expressed in E. coli B834(DE3) strain using LeMaster media and purified with the same protocol as the native protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18376</offset><text>Protein crystallization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18400</offset><text>Crystals of the native SPOC domain of FPA were grown at 20°C with the sitting-drop vapor diffusion method. The protein solution was at 30 mg/ml concentration, and the reservoir solution contained 0.2 M MgSO4, and 20% (v/v) PEG 3350. Fully-grown crystals were obtained two days after set-up. Crystals of the selenomethionine labeled SPOC domain were grown using the same condition as the native protein. The crystals were cryo-protected in the crystallization solution supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen for data collection at 100K.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18971</offset><text>Data collection and processing</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19002</offset><text>A single-wavelength anomalous dispersion (SAD) X-ray diffraction data set on a selenomethionine labeled SPOC domain crystal was collected at the National Synchrotron Light Source (NSLS) beamline X29A using an ADSC Q315r CCD. The diffraction images were processed and scaled with the HKL package. The crystal belongs to space group P65, with unit cell parameters of a = b = 108.2 Å, and c = 34.2 Å.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>19402</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>19441</offset><text>The structure of the SPOC domain was solved by the selenomethionyl SAD method with the program SHELX. The phases were used by program PHENIX for automatic model building. Manual model rebuilding was carried out with Coot. The structure refinement was performed with the program PHENIX, with translation, libration, and screw-rotation (TLS) parameters. The data processing and refinement statistics are summarized in Table 1. The Ramachandran plot showed that 95.8% of the residues are located in the most favored regions, and 4.2% are in additional allowed regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20007</offset><text>Generation of constructs with mutated genomic FPA sequence</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20066</offset><text>A series of constructs containing a mutated FPA genomic sequence was prepared based on pGreen I 0029 vector. pGreen I 0029 vector with inserted FPA genomic sequence was prepared. In this vector FPA genomic sequence is flanked by 2620bp of the native sequence upstream to the start codon and 1178bp downstream to the stop codon. The vector contains kanamycin resistance genes for both the bacteria and plant hosts. In order to obtain a series of constructs with mutated FPA genomic sequence, FPA sequence in this construct was modified using site-directed mutagenesis. Primers used to prepare required constructs are listed in S1 Table. After the mutagenesis reaction the presence of only the desired mutations was confirmed by sequencing of the whole FPA genomic sequence and flanking regions.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20860</offset><text>Generation of Arabidopsis thaliana transgenic plants</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20913</offset><text>All transgenic plants were prepared in fpa-8 mutant background, which is in Col-0 accession. The prepared vectors for Arabidopsis transformations were introduced into electro-competent Agrobacterium tumefaciens cells (C58 CV3101 strain harbouring pSoup vector). The floral dip method was used for plant transformation. Transgenic plants were selected using kanamycin as a selection marker. Presence of the desired mutations in plants was confirmed with specific dCaps markers.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>21390</offset><text>Plant growth conditions</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21414</offset><text>Wild type Col-0 plants used in this study were obtained from the Nottingham Arabidopsis Stock Centre. Seed of fpa-8 and 35S::FPA:YFP were obtained from Professor Caroline Dean. Plants were grown in pots containing Universal Extra general purpose soil. The glasshouse temperature was maintained at 20°C and the 16 hour daylight was provided by high pressure sodium vapour lamps (Philips Powertone SON-T AGRO 400). In order to grow plants in sterile conditions, seeds were first surface sterilized by a 5 min treatment with sterilizing solution (3% v/v sodium hypochlorite, 0.02% v/v Triton X-100), followed by three washes with 0.02% v/v Triton X-100 and one wash with sterile water. The sterile seeds were sown on MS10 media supplemented with 0.8% w/v agar. MS10 medium was also supplemented with specific antibiotics if required. After sowing, the seeds were stratified at 4°C for two days in order to synchronize their germination. Plants were grown in the tissue culture room at the following conditions: temperature 22°C, 16 hours daylight provided by the Master TL-D 36W/840 (Philips) lamps.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22514</offset><text>Plant protein analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22537</offset><text>Total protein samples were prepared using extraction buffer containing: 40 mM Tris-HCl, pH 6.8; 0.1 mM EDTA, pH 8.0; 8 M urea; 1.43 M β-mercaptoethanol, 7% v/v Complete Protease Inhibitors (Roche) and 5 mM PMSF. Equal volumes of samples were separated on 8% SDS-PAGE. Proteins were transferred onto Protran nitrocellulose transfer membrane (Whatman) using wet Criterion blotter system (BioRad). The transfer was performed at room temperature for two hours at a stable voltage of 70 V. Membrane was blocked in 3% (w/v) Milk in TBS for 1h at room temperature followed by overnight incubation with anti-FPA antibody (dilution 1:100 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) (1:3000 dilution in 3% (w/v) Milk in TBS). Protein was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). Blots were re-probed following treatment with low pH solution (25mM glycine-HCl, pH 2, 1% (w/v) SDS) followed by blocking for 1h at room temperature in 3% (w/v) Milk in TBS. The membrane was incubated overnight with anti-TUBB2A, tubulin, beta 2A antibody (ARP40177_P050 Aviva systems biology; (dilution 1:1000 in 3% (w/v) Milk in TBS). After washes the membrane was incubated for 75 min with goat anti-rabbit antibody (Thermo Scientific) [1:3000 dilution in 3% (w/v) Milk in TBS]. Signal was detected using SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>24024</offset><text>RNA gel blot analysis and RT-qPCR</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>24058</offset><text>RNA gel blot analysis and RT-qPCR method performed as previously described.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>24134</offset><text>Supporting Information</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>24157</offset><text>References</text></passage><passage><infon key="fpage">688</infon><infon key="lpage">700</infon><infon key="name_0">surname:Moore;given-names:MJ</infon><infon key="name_1">surname:Proudfoot;given-names:NJ</infon><infon 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<collection><source>PMC</source><date>20201219</date><key>pmc.key</key><document><id>4993997</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/srep31425</infon><infon key="article-id_pii">srep31425</infon><infon key="article-id_pmc">4993997</infon><infon key="article-id_pmid">27550639</infon><infon key="elocation-id">31425</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Chen;given-names:Ji-Yun</infon><infon key="name_1">surname:Liu;given-names:Liang</infon><infon key="name_2">surname:Cao;given-names:Chun-Ling</infon><infon key="name_3">surname:Li;given-names:Mei-Jun</infon><infon key="name_4">surname:Tan;given-names:Kemin</infon><infon key="name_5">surname:Yang;given-names:Xiaohan</infon><infon key="name_6">surname:Yun;given-names:Cai-Hong</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">6</infon><infon key="year">2016</infon><offset>0</offset><text>Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>96</offset><text>N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1386</offset><text>Acetylation is one of the most ubiquitous modifications that plays a vital role in many biological processes, such as transcriptional regulation, protein-protein interaction, enzyme activity, protein stability, antibiotic resistance, biological rhythm and so on. Protein acetylation can be grouped into lysine Nε-acetylation and peptide N-terminal acetylation (Nt-acetylation). Generally, Nε-acetylation refers to the transfer of an acetyl group from an acetyl coenzyme A (Ac-CoA) to the ε-amino group of lysine. This kind of modification is catalyzed by lysine acetyltransferases (KATs), some of which are named histone acetyltransferases (HATs) because early studies focused mostly on the post-transcriptional acetylation of histones.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2141</offset><text>Despite the prominent accomplishments in the field regarding Nε-acetylation by KATs for over 50 years, the significance of the more evolutionarily conserved Nt-acetylation is still inconclusive. Nt-acetylation is an abundant and evolutionarily conserved modification occurring in bacteria, archaea and eukaryotes. It is estimated that about 80–90% of soluble human proteins and 50–70% of yeast proteins are subjected to Nt-acetylation, where an acetyl moiety is transferred from Ac-CoA to the α-amino group of the first residue. Recently Nt-acetylome expands the Nt-acetylation to transmembrane proteins. Unlike Nε-acetylation that can be eliminated by deacetylases, Nt-acetylation is considered irreversible since no corresponding deacetylase is found to date. Although Nt-acetylation has been regarded as a co-translational modification traditionally, there is evidence that post-translational Nt-acetylation exists. During the past decades, a large number of Nt-acetylome researches have shed light on the functional roles of Nt-acetylation, including protein degradation, subcellular localization, protein-protein interaction, protein-membrane interaction, plant development, stress-response and protein stability.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3376</offset><text>The Nt-acetylation is carried out by N-terminal acetyltransferases (NATs) that belong to the GNAT superfamily. To date, six NATs (NatA/B/C/D/E/F) have been identified in eukaryotes. About 40 percent of Nt-acetylation of soluble proteins in cells is catalyzed by NatA complex which is composed of the catalytic subunit Naa10p and the auxiliary subunit Naa15p. NatE was found to physically interact with the NatA complex without any observation of impact on NatA-activity. Two other multimeric complexes of NATs are NatB and NatC which contain the catalytic subunits Naa20 and Naa30 and the auxiliary subunits Naa25 and Naa35/Naa38, respectively. Furthermore, only the catalytic subunits Naa40 and Naa60 were found for NatD and NatF, respectively. Besides Nt-acetylation, accumulating reports have proposed Nε-acetylation carried out by NATs.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4223</offset><text>There is an evolutionary increasing in the degree of Nt-acetylation between yeast and human which could partly be explained by the contribution of NatF. As the first N-terminal acetyltransferase discovered on an organelle, NatF, encoded by NAA60 and also named as Histone acetyltransferase type B protein 4 (HAT4), Naa60 or N-acetyltransferase 15 (NAT15), is the youngest member of the NAT family. Unlike other NATs that are highly conserved among lower and higher eukaryotes, NatF only exists in higher eukaryotes. Subsequent researches indicated that NatF displays its catalytic ability with both Nt-acetylation and lysine Nε-acetylation. As an N-terminal acetyltransferase, NatF can specifically catalyze acetylation of the N-terminal α-amine of most transmembrane proteins and has substrate preference towards proteins with Met-Lys-, Met-Val-, Met-Ala- and Met-Met-N-termini, thus partially overlapping substrate selectivity with NatC and NatE. On the other hand, NatF, with its lysine acetyltransferase activity, mediates the lysine acetylation of free histone H4, including H4K20, H4K79 and H4K91. Another important feature of NatF is that this protein is anchored on the Golgi apparatus through its C-terminal membrane-integrating region and takes part in the maintaining of Golgi integrity. With its unique intracellular organellar localization and substrate selectivity, NatF appears to provide more evolutionary information among the NAT family members.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5697</offset><text>It was recently found that NatF facilitates nucleosomes assembly and that NAA60 knockdown in MCF7-cell inhibits cell proliferation, sensitizes cells to DNA damage and induces cell apoptosis. In Drosophila cells, NAA60 knockdown induces chromosomal segregation defects during anaphase including lagging chromosomes and chromosomal bridges. Much recent attention has also been focused on the requirement of NatF for regulation of organellar structure. In HeLa cells, NAA60 knockdown causes Golgi apparatus fragmentation which can be rescued by overexpression Naa60. The systematic investigation of publicly available microarray data showed that NATs share distinct tissue-specific expression patterns in Drosophila and NatF shows a higher expression level in central nervous system of Drosophila.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6492</offset><text>In this study, we solved the structures of human Naa60 (NatF) in complex with coenzyme. The hNaa60 protein contains a unique amphipathic α-helix (α5) following its GNAT domain that might account for the Golgi localization of this protein. Crystal structures showed that the β7-β8 hairpin rotated about 50 degrees upon removing the C-terminal region of the protein and this movement substantially changed the geometry of the substrate-binding pocket. Remarkably, we find that Phe 34 may participate in the proper positioning of the coenzyme for the transfer reaction to occur. Further structure comparison and biochemical studies also identified other key structural elements essential for the enzyme activity of Naa60.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>7225</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>7233</offset><text>Overall structure of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7261</offset><text>In the effort to prepare the protein for structural studies, we tried a large number of hNaa60 constructs but all failed due to heavy precipitation or aggregation. Sequence alignment of Naa60 from different species revealed a Glu-Glu-Arg (EER) versus Val-Val-Pro (VVP) sequence difference near the N-terminus of the protein in Xenopus Laevis versus Homo sapiens (Fig. 1A). Considering that terminal residues may lack higher-order structure and hydrophobic residues in this region may expose to solvent and hence cause protein aggregation, we mutated residues 4–6 from VVP to EER for the purpose of improving solubility of this protein. According to previous studies, this N-terminal region should not interfere with hNaa60’s Golgi localization. We tried many hNaa60 constructs with the three-residues mutation but only the truncated variant 1-199 and the full-length protein behaved well. We obtained the crystal of the truncated variant 1-199 in complex with CoA first, and after extensive trials we got the crystal of the full-length protein (spanning residues 1-242) in complex with Ac-CoA (Fig. 1B,C). Hereafter, all deletions or point mutants of hNaa60 we describe here are with the EER mutation. The crystal structures of hNaa60(1-242)/Ac-CoA and hNaa60(1-199)/CoA were determined by molecular replacement and refined to 1.38 Å and 1.60 Å resolution, respectively (Table 1). The electron density maps were of sufficient quality to trace residues 1-211 of hNaa60(1-242) and residues 5-199 of hNaa60(1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8783</offset><text>The structure of hNaa60 protein contains a central domain exhibiting a classic GCN5-related N-acetyltransferase (GNAT) folding, along with the extended N- and C-terminal regions (Fig. 1B,C). The central domain includes nine β strands (β1-β9) and four α-helixes (α1-α4) and is highly similar to the known hNaa50p and other reported NATs (Fig. 1D). However, in hNaa60, there is an extra 20-residue loop between β3 and β4 that forms a small subdomain with well-defined 3D structure (Fig. 1B–D). Furthermore, the β7-β8 strands form an approximately antiparallel β-hairpin structure remarkably different from that in hNaa50p (Fig. 1D). The N- and C-terminal regions form helical structures (α0 and α5) stretching out from the central GCN5-domain (Fig. 1C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9579</offset><text>Interestingly, we found that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) (Figure S1), indicating that residues 200–242 may have some auto-inhibitory effect on the activity of the enzyme. However, since this region was not visible in the hNaa60(1-242) crystal structure, we do not yet understand how this happens. Another possibility is that since hNaa60 is localized on Golgi apparatus, the observed low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies. For the convenience of studying the kinetics of mutants, the mutagenesis studies described hereafter were all based on hNaa60 (1-199).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10278</offset><text>An amphipathic α-helix in the C-terminal region may contribute to Golgi localization of hNaa60</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10377</offset><text>There is one hNaa60 molecule in the asymmetric unit in the hNaa60(1-242)/Ac-CoA structure. The C-terminal region extended from the GCN5-domain forms an amphipathic helix (α5) and interacts with a molecule in a neighbor asymmetric unit through hydrophobic interactions between α5-helix and a hydrophobic groove between the N-terminal β1 and β3 strands of the neighbor molecule (Fig. 2A). The C-terminal extension following α5-helix forms a β-turn that wraps around and interacts with the neighbor protein molecule through hydrophobic interactions, too. In the hNaa60(1-199)/CoA structure, a part of the α5-helix is deleted due to truncation of the C-terminal region (Fig. 1B). Interestingly, the remaining residues in α5-helix still form an amphipathic helix although the hydrophobic interaction with the N-terminal hydrophobic groove of a neighbor molecule is abolished and the helix is largely exposed in solvent due to different crystal packing (Fig. 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11364</offset><text>A recent research showed that residues 182–216 are important for the localization of hNaa60 on Golgi. According to our structure, the solvent-exposed amphipathic helix (α5) formed by residues 190-202 with an array of hydrophobic residues located on one side (Ile 190, Leu 191, Ile 194, Leu 197 and Leu 201) and hydrophilic residues on the other side (Fig. S2) might account for interaction between hNaa60 and Golgi membrane, as it is a typical structure accounting for membrane association through immersing into the lipid bi-layer with its hydrophobic side as was observed with KalSec14, Atg3, PB1-F2 etc.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>11975</offset><text>The β7-β8 hairpin showed alternative conformations in the hNaa60 crystal structures</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12065</offset><text>Superposition of hNaa60(1-242)/Ac-CoA, hNaa60(1-199)/CoA and hNaa50/CoA/peptide (PDB 3TFY) revealed considerable difference in the β7-β8 hairpin region despite the overall stability and similarity of the GNAT domain (Fig. 1D). In hNaa60(1-242), the β7-β8 hairpin is located in close proximity to the α1-α2 loop, creating a more compact substrate binding site than that in hNaa50, where this region adopts a more flexible loop conformation (β6-β7 loop). Upon removing the C-terminal region of hNaa60, we observed that hNaa60 (1-199) molecules pack in a different way involving the β7-β8 hairpin in the crystal, leading to about 50 degree rotation of the hairpin which moves away from the α1-α2 loop (Figs 1D and 2C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12821</offset><text>This conformational change substantially altered the geometry of the substrate binding site, which could potentially change the way in which the substrate accesses the active site of the enzyme. In hNaa60(1-242), the β7-β8 hairpin covers the active site in a way similar to that observed in hNaa50, presumably leaving only one way for the substrate to access the active site, i.e. to enter from the opposite end into the same tunnel where Ac-CoA/CoA binds (Fig. 2D), which may accommodate access of a NAT substrate only. KAT activity of hNaa60 toward histone H4 has been noted in previous study, and our enzyme kinetic data also indicated that hNaa60 can acetylate H3-H4 tetramer in vitro (Figure S3). Furthermore, we analyzed the acetylation status of histone H3-H4 tetramer using mass spectrometry and observed that multiple lysine residues in the protein showed significantly increased acetylation level and changed acetylation profile upon treatment with hNaa60(1-199) (Figure S4). We also conducted liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis on a synthetic peptide (NH2-MKGKEEKEGGAR-COOH) after treatment with hNaa60(1-199), and the data confirmed that both the N-terminal α-amine and lysine side-chain ε-amine were robustly acetylated after the treatment (Table S1). Despite these observations, the mechanism for this alternative activity remains unknown. Recent structural investigation of other NATs proposed that the β6-β7 loop, corresponding to the β7-β8 hairpin in hNaa60, and the α1-α2 loop flanking the substrate-binding site of NATs, prevent the lysine side-chain of the KAT substrates from inserting into the active site. Indeed, superposition of hNaa60(1-242) structure on that of Hat1p, a typical KAT, in complex with a histone H4 peptide revealed obvious overlapping/clashing of the H4 peptide (a KAT substrate) with the β7-β8 hairpin of hNaa60(1-242) (Fig. 2D). Interestingly, in the hNaa60(1-199) crystal structure, the displaced β7-β8 hairpin opened a second way for the substrate to access the active center that would readily accommodate the binding of the H4 peptide (Fig. 2E), thus implied a potential explanation for KAT activity of this enzyme from a structural biological view. However, since hNaa60(1-242) and hNaa60(1-199) were crystallized in different crystal forms, the observed conformational change of the β7-β8 hairpin may simply be an artifact related to the different crystal packing. Whether the KAT substrates bind to the β7-β8 hairpin displaced conformation of the enzyme needs to be verified by further structural and functional studies.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15485</offset><text>Phe 34 facilitates proper positioning of the cofactor for acetyl-transfer</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15559</offset><text>The electron density of Phe 34 side-chain is well defined in the hNaa60(1-242)/Ac-CoA structure, but becomes invisible in the hNaa60(1-199)/CoA structure, indicating displacement of the Phe 34 side-chain in the latter (Fig. 3A,B). A solvent-derived malonate molecule is found beside Phe 34 and the ethanethioate moiety of Ac-CoA in the high-resolution hNaa60(1-242)/Ac-CoA structure (Fig. 3A). Superposition of this structure on that of hNaa50p/CoA/peptide shows that the malonate molecule overlaps well on the N-terminal methionine of the substrate peptide and residue Phe 34 in hNaa60 overlaps well on Phe 27 in hNaa50 (Fig. 4A). Interestingly, in the structure of hNaa60(1-199)/CoA, the terminal thiol of CoA adopts alternative conformations. One is to approach the substrate amine (as indicated by the superimposed hNaa50/CoA/peptide structure), similar to the terminal ethanethioate of Ac-CoA in the structure of hNaa60(1-242)/Ac-CoA; the other is to approach the α1-α2 loop and away from the substrate amine (Fig. 3B). To rule out the possibility that the electron density we define as the alternative conformation of the thiol terminus is residual electron density of the displaced side-chain of Phe 34, we solved the crystal structure of hNaa60(1-199) F34A/CoA. The structure of this mutant is highly similar to hNaa60(1-199)/CoA and there is essentially the same electron density corresponding to the alternative conformation of the thiol (Fig. 3C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17026</offset><text>Phe 27 in hNaa50p (equivalent to Phe 34 in hNaa60) has been implicated to facilitate the binding of N-terminal methionine of the substrate peptide through hydrophobic interaction. However, in the hNaa60/Ac-CoA structure, a hydrophilic malonate molecule is found at the same location where the N-terminal methionine should bind as is indicated by the superposition (Fig. 3A), suggesting that Phe 34 may accommodate binding of hydrophilic substrate, too. Moreover, orientation of Phe 34 side-chain seems to be co-related to positioning of the terminus of the co-enzyme and important for placing it at a location in close proximity to the substrate amine. We hypothesize that if Phe 34 only works to facilitate the binding of the hydrophobic N-terminal Met residue, to mutate it from Phe to Ala would not abolish the catalytic activity of this enzyme, while if Phe 34 also plays an essential role to position the ethanethioate moiety of Ac-CoA, the mutation would be expected to abrogate the activity of the enzyme. Indeed, our enzyme kinetic data showed that hNaa60(1-199) F34A mutant showed no detectable activity (Fig. 5A). In order to rule out the possibility that the observed loss of activity may be related to bad folding of the mutant protein, we studied the circular dichroism (CD) spectrum of the protein (Fig. 5B) and determined its crystal structure (Fig. 3C). Both studies proved that the F34A mutant protein is well-folded. Many studies have addressed the crucial effect of α1-α2 loop on catalysis, showing that some residues located in this area are involved in the binding of substrates. We propose that Phe 34 may play a dual role both in interacting with the peptide substrate (recognition) and in positioning of the ethanethioate moiety of Ac-CoA to the right location to facilitate acetyl-transfer.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18850</offset><text>Structural basis for hNaa60 substrate binding</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18896</offset><text>Several studies have demonstrated that the substrate specificities of hNaa60 and hNaa50 are highly overlapped. The structure of hNaa50p/CoA/peptide provides detailed information about the position of substrate N-terminal residues in the active site of hNaa50. Comparing the active site of hNaa60(1-242)/Ac-CoA with hNaa50p/CoA/peptide revealed that key catalytic and substrate binding residues are highly conserved in both proteins (Fig. 4A). With respect to catalysis, hNaa50p has been shown to employ residues Tyr 73 and His 112 to abstract proton from the α-amino group from the substrate’s first residue through a well-ordered water. A well-ordered water was also found between Tyr 97 and His 138 in hNaa60 (1-199)/CoA and hNaa60 (1-242)/Ac-CoA (Fig. 4B). To determine the function of Tyr 97 and His 138 in hNaa60 catalysis, we mutated these residues to alanine and phenylalanine, respectively, and confirmed that all these mutants used in our kinetic assays are well-folded by CD spectra (Fig. 5B). Purity of all proteins were also analyzed by SDS-PAGE (Figure S5). As show in Fig. 5A, the mutants Y97A, Y97F, H138A and H138F abolished the activity of hNaa60. In contrast, to mutate the nearby solvent exposed residue Glu 37 to Ala (E37A) has little impact on the activity of hNaa60 (Figs 4B and 5A). In conclusion, the structural and functional studies indicate that hNaa60 applies the same two base mechanism through Tyr 97, His 138 and a well-ordered water as was described for hNaa50.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20394</offset><text>The malonate molecule observed in the hNaa60(1-242)/Ac-CoA crystal structure may be indicative of the substrate binding position of hNaa60 since it is located in the active site and overlaps the N-terminal Met of the substrate peptide in the superposition with the hNaa50p/CoA/peptide structure (Fig. 4A). Residues Tyr 38, Asn 143 and Tyr 165 are located around the malonate and interact with it through direct hydrogen bonds or water bridge (Fig. 4C). Although malonate is negatively charged, which is different from that of lysine ε-amine or peptide N-terminal amine, similar hydrophilic interactions may take place when substrate amine presents in the same position, since Tyr 38, Asn 143 and Tyr 165 are not positively or negatively charged. In agreement with this hypothesis, it was found that the Y38A, N143A and Y165A mutants all showed remarkably reduced activities as compared to WT, implying that these residues may be critical for substrate binding (Figs 4C and 5A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21378</offset><text>The β3-β4 loop participates in the regulation of hNaa60-activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21449</offset><text>Residues between β3 and β4 of hNaa60 form a unique 20-residue long loop (residues 73–92) that is a short turn in many other NAT members (Fig. 1D). Previous study indicated that auto-acetylation of hNaa60K79 could influence the activity of hNaa60; however, we were not able to determine if Lys 79 is acetylated in our crystal structures due to poor quality of the electron density of Lys 79 side-chain. We therefore used mass spectrometry to analyze if Lys 79 was acetylated in our bacterially purified proteins, and observed no modification on this residue (Figure S6). To assess the impact of hNaa60K79 auto-acetylation, we studied the kinetics of K79R and K79Q mutants which mimic the un-acetylated and acetylated form of Lys 79, respectively. Interestingly, both K79R and K79Q mutants led to an increase in the catalytic activity of hNaa60, while K79A mutant led to modest decrease of the activity (Fig. 5A). These data indicate that the acetylation of Lys 79 is not required for optimal catalytic activity of hNaa60 in vitro.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22485</offset><text>It is noted that the β3-β4 loop of hNaa60 acts like a door leaf to partly cover the substrate-binding pathway. We hence hypothesize that the β3-β4 loop may interfere with the access of the peptide substrates and that the solvent-exposing Lys 79 may play a potential role to remove the door leaf when it hovers in solvent (Fig. 4D). Acidic residues Glu 80, Asp 81 and Asp 83 interact with His 138, His 159 and His 158 to maintain the conformation of the β3-β4 loop, thus contribute to control the substrate binding (Fig. 4D). To verify this hypothesis, we mutated Glu 80, Asp 81 and Asp 83 to Ala respectively. In line with our hypothesis, E80A, D81A and D83A mutants exhibit at least 2-fold increase in hNaa60-activity (Fig. 5A). Interestingly, the structure of an ancestral NAT from S. solfataricus also exhibits a 10-residue long extension between β3 and β4, and the structure and biochemical studies showed that the extension of SsNat has the ability to stabilize structure of the active site and potentiate SsNat-activity.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>23536</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>23547</offset><text>Nt-acetylation, which is carried out by the NAT family acetyltransferases, is an ancient and essential modification of proteins. Although many NATs are highly conserved from lower to higher eukaryotes and the substrate bias of them appears to be partially overlapped, there is a significant increase in the overall level of N-terminal acetylation from lower to higher eukaryotes. In this study we provide structural insights into Naa60 found only in multicellular eukaryotes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>24023</offset><text>The N-terminus of hNaa60 harbors three hydrophobic residues (VVP) that makes it very difficult to express and purify the protein. This problem was solved by replacing residues 4–6 from VVP to EER that are found in Naa60 from Xenopus Laevis. Since Naa60 from human and from Xenopus Laevis are highly homologous (Fig. 1A), we speculate that these two proteins should have the same biological function. Therefore it is deduced that the VVP to EER replacement on the N-terminus of hNaa60 may not interfere with its function. However, in the hNaa60(1-242) structure the N-terminus adopts an α-helical structure which will probably be kinked if residue 6 is proline (Fig. 1C), and in the hNaa60(1-199) structure the N-terminus adopts a different semi-helical structure (Fig. 1B) likely due to different crystal packing. Hence it is not clear if the N-terminal end of wild-type hNaa60 is an α-helix, and what roles the hydrophobic residues 4–6 play in structure and function of wild-type hNaa60. In addition to the three-residue mutation (VVP to EER), we also tried many other hNaa60 constructs, but only the full-length protein and the truncated variant 1-199 behaved well. The finding that the catalytic activity of hNaa60(1-242) is much lower than that of hNaa60(1-199) is intriguing. We speculate that low activity of the full-length hNaa60 might be related to lack of Golgi localization of the enzyme in our in vitro studies or there remains some undiscovered auto-inhibitory regulation in the full-length protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25544</offset><text>The hNaa60 protein was proven to be localized on Golgi apparatus. Aksnes and colleagues predicted putative transmembrane domains and two putative sites of S-palmitoylation, by bioinformatics means, to account for Golgi localization of the protein. They then mutated all five cysteine residues of hNaa60’s to serine, including the two putative S-palmitoylation sites. However, these mutations did not abolish Naa60 membrane localization, indicating that S-palmitoylation is unlikely to (solely) account for targeting hNaa60 on Golgi. Furthermore, adding residues 217–242 of hNaa60 (containing residues 217–236, one of the putative transmembrane domains) to the C terminus of eGFP were not sufficient to localize the protein on Golgi apparatus, while eGFP-hNaa60182-242 was sufficient to, suggesting that residues 182–216 are important for Golgi localization of hNaa60. We found that residues 190–202 formed an amphipathic helix with an array of hydrophobic residues located on one side. This observation is reminiscent of the protein/membrane interaction through amphipathic helices in the cases of KalSec14, Atg3, PB1-F2 etc. In this model an amphipathic helix can immerse its hydrophobic side into the lipid bilayer through hydrophobic interactions. Therefore we propose that the amphipathic helix α5 may contribute to Golgi localization of hNaa60. This model, though may need further studies, is supported by the Aksnes studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>26986</offset><text>Previous studies indicated that members of NAT family are bi-functional NAT and KAT enzymes. However, known structures of NATs do not well support this hypothesis, since the β6-β7 hairpin/loop of most of NATs is involved in the formation of a tunnel-like substrate-binding site with the α1-α2 loop, which would be good for the NAT but not KAT activity of the enzyme. Kinetic studies have been conducted to compare the NAT and KAT activity of hNaa50 in vitro, and indicate that the NAT activity of Naa50 is much higher than KAT activity. However, the substrate used in this study for assessing KAT activity was a small peptide which could not really mimic the 3D structure of a folded protein substrate in vivo. Our mass spectrometry data indicated that there were robust acetylation of histone H3-H4 tetramer lysines and both N-terminal acetylation and lysine acetylation of the peptide used in the activity assay, thus confirmed the KAT activity of this enzyme in vitro. Conformational change of the β7-β8 hairpin (corresponding to the β6-β7 loop of other NATs) is noted in our structures (Figs 1D and 2C), which might provide an explanation to the NAT/KAT dual-activity in a structural biological view, but we were unable to rule out the possibility that the observed conformational change of this hairpin might be an artifact related to crystal packing or truncation of the C-terminal end of the protein. Further studies are therefore needed to reveal the mechanism for the KAT activity of this enzyme.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28518</offset><text>The relationship between enzyme, co-enzyme and substrates has been documented for several years. In early years, researchers found adjustment of GCN5 histone acetyltransferase structure when it binds CoA molecule. The complexed form of NatA is more suitable for catalytic activation, since the α1-α2 loop undergoes a conformation change to participate in the formation of substrate-binding site when the auxiliary subunit Naa15 interacts with Naa10 (the catalytic subunit of NatA). In the structure of hNaa50/CoA/peptide, Phe 27 in the α1-α2 loop appears to make hydrophobic interaction with the N-terminal Met of substrate peptide. However, the hNaa60(1-242)/Ac-CoA crystal structure indicated that its counterpart in hNaa60, Phe 34, could also accommodate the binding of a hydrophilic malonate that occupied the substrate binding site although it maintained the same conformation as that observed in hNaa50. Interestingly, the terminal thiol of CoA adopted alternative conformations in the structure of hNaa60(1-199)/CoA. One was to approach the substrate amine; the other was to approach the α1-α2 loop and away from the substrate amine. Same alternative conformations of CoA were observed in the hNaa60(1-199)(F34A) crystal structure, and our kinetic data showed that the F34A mutation abolished the activity of the enzyme. Taken together, our data indicated that Phe 34 in hNaa60 may play a role in placing co-enzyme at the right location to facilitate the acetyl-transfer. However, these data did not rule out that possibility that Phe 34 may coordinate the binding of the N-terminal Met through hydrophobic interaction as was proposed by previous studies.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30205</offset><text>Furthermore, we showed that hNaa60 adopts the classical two base mechanism to catalyze acetyl-transfer. Although sequence identity between hNaa60 and hNaa50 is low, key residues in the active site of both enzymes are highly conserved. This can reasonably explain the high overlapping substrates specificities between hNaa60 and hNaa50. Another structural feature of hNaa60 that distinguishes it from other NATs is the β3-β4 long loop which appears to inhibit the catalytic activity of hNaa60. However, this loop also seems to stabilize the whole hNaa60 structure, because deletion mutations of this region led to protein precipitation and aggregation (Figure S7). A previous study suggested that the auto-acetylation of Lys 79 was important for hNaa60-activity, whereas the point mutation K79R did not decrease the activity of hNaa60 in our study. Meanwhile, no electron density of acetyl group was found on Lys 79 in our structures and mass spectrometry analysis. Hence, it appears that the auto-acetylation of hNaa60 is not an essential modification for its activity for the protein we used here. As for the reason why K79R in Yang’s previous studies reduced the activity of the enzyme, but in our studies it didn’t, we suspect that the stability of this mutant may play some role. K79R is less stable than the wild-type enzyme as was judged by its poorer gel-filtration behavior and tendency to precipitate. In our studies we have paid special attention and carefully handled this protein to ensure that we did get enough of the protein in good condition for kinetic assays. The intracellular environment is more complicated than our in vitro assay and the substrate specificity of hNaa60 most focuses on transmembrane proteins. The interaction between hNaa60 and its substrates may involve the protein-membrane interaction which would further increase the complexity. It is not clear if the structure of hNaa60 is different in vivo or if other potential partner proteins may help to regulate its activity. Nevertheless, our study may be an inspiration for further studies on the functions and regulation of this youngest member of the NAT family.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>32362</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32370</offset><text>Cloning, expression and purification of Homo sapiens Naa60 (hNaa60)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32438</offset><text>The cDNA encoding hNaa60 residues 1–242 (full-length) or residues 1–199 were amplified by PCR and inserted into the pET23a vector, which had been modified to provide an N-terminal 6xHis-tag followed by a tobacco etch virus (TEV) protease cleavage site. The VVP to EER (residues 4–6) mutation and other mutations for functional studies were introduced using the quick change method. The protein was expressed in Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS at 16 °C for 15 h in the presence of 0.1 mM IPTG. Cells were harvested at 4 °C by centrifugation (4,000 g for 10 min) and resuspended in buffer A containing 20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10% glycerol, 1 mM protease inhibitor PMSF (Phenylmethylsulfonyl fluoride) and 1 mM Tris (2-carboxyethyl)phosphine (TCEP) hydrochloride. Cells were lysed by sonication and the lysate was cleared by centrifugation (18,000 g at 4 °C for 20 min). Then the supernatant was loaded onto a 5-mL Chelating Sepharose column (GE Healthcare) charged with Ni2+ and washed with buffer B (20 mM Tris, pH 8.0, 500 mM NaCl, 50 mM imidazole, 1% glycerol and 1 mM TCEP). The protein was eluted with buffer C (20 mM Tris, pH 8.0, 500 mM NaCl, 300 mM imidazole, 1% glycerol and 1 mM TCEP). The eluent was digested by His-tagged TEV protease and concentrated by ultrafiltration at the same time. After 3 hours, the concentrated eluent was diluted 10 times with buffer D (20 mM Tris, pH 8.0, 500 mM NaCl, 1% glycerol and 1 mM TCEP) and the diluent was passed through the nickel column once again to remove the His-tagged TEV protease and the un-cleaved His-hNaa60 protein. The flow-through was concentrated to 500 μl and loaded onto a Superose 6 or Superdex 200 10/300 gel-filtration column (GE Healthcare) equilibrated with buffer E (20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP). Fractions containing the protein were collected and concentrated to a final concentration of 10 mg/ml for crystallization or acetyltransferases assays.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34517</offset><text>Circular Dichroism (CD) Spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34554</offset><text>CD spectra of the proteins were obtained using a Jasco J-810 circular dichroism spectropolarimeter scanning from 190 to 250 nm with a 1 mm quartz cuvette. The wild-type and mutant proteins were examined at 4.5 μM concentration in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature. All samples were centrifuged at 10,000 g for 5 min before analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34949</offset><text>Crystallization, data collection and structure determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35010</offset><text>The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35814</offset><text>The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000. The hNaa60(1-199) structure was determined by molecular replacement with Phaser using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP in CCP4 or simulated-annealing in CNS were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT and Phenix. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36891</offset><text>Acetyltransferase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36915</offset><text>Acetyltransferase assay of hNaa60 was conducted as described previously. Briefly, a reaction cocktail containing 100 mM Tris-HCl buffer, pH 8.5, 0.07% alkylated BSA, 0.01% NP-40, 1 mM EDTA, 150 μM Ac-CoA (Sigma) was prepared and varied concentrations of the substrate peptide (0–400 μM) (NH2-MKGKEEKEGGAR-COOH) was added in a 1.5-mL microfuge tube, and then the respective enzyme was added to initiate the reaction with a final assay volume of 100 μL. The reaction was carried out for 20 minutes at 37 °C. Aliquots (40 μL) of the reaction were then removed and quenched with 40 μL of ice-cold isopropanol in individual wells of a 96-well black microplate (Corning), and then mixed with 80 μl of 25 μM 7-diethylamino-3-(49 maleimidylphenyl)-4-methylcoumarin (CPM) (Sigma) in 100 mM Tris-HCl (pH 8.5) and 1% Triton X-100 and allowed to react in darkness for 10 minutes prior to reading. The fluorescence signal was monitored using a Varioskan Flash plate reader (Thermo Scientific) at Exmax = 385 nm and Emmax = 465 nm. Substrate inhibition appeared at high concentrations of substrate peptide prevented our kinetics assays from reaching saturation of the enzyme. Therefore, we determined the value of kcat/Km by fitting our data to the equation: v = (kcat/Km)[ET][S] when the substrate concentration was far less than Km. The assays were done in triplicate. The slope of the line indicates the kcat/Km value of the enzyme (Figure S1).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>38401</offset><text>Additional Information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38424</offset><text>How to cite this article: Chen, J.-Y. et al. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase. Sci. 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J.-Y.C. and C.-H.Y. wrote the manuscript.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>42521</offset><text>Overall structure of Naa60.</text></passage><passage><infon key="file">srep31425-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>42549</offset><text>(A) Sequence alignment of Naa60 (NatF, HAT4) from different species including Homo sapiens (Homo), Bos mutus (Bos), Salmo salar (Salmo) and Xenopus (Silurana) tropicalis (Xenopus). Alignment was generated using NPS@ and ESPript.3.0 (http://espript.ibcp.fr/ESPript/ESPript/). Residues 4–6 are highlighted in red box. (B) The structure of hNaa60(1-199)/CoA complex is shown as a yellow cartoon model. The CoA molecule is shown as sticks. (C) The structure of hNaa60(1-242)/Ac-CoA complex is presented as a cartoon model in cyan. The Ac-CoA and malonate molecules are shown as cyan and purple sticks, respectively. The secondary structures are labeled starting with α0. (D) Superposition of hNaa60(1-242) (cyan), hNaa60(1-199) (yellow) and hNaa50 (pink, PDB 3TFY). The Ac-CoA of hNaa60(1-242)/Ac-CoA complex is represented as cyan sticks.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43389</offset><text>Amphipathicity of the α5 helix and alternative conformations of the β7-β8 hairpin.</text></passage><passage><infon key="file">srep31425-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43482</offset><text>(A) The α5 helix of hNaa60(1-242) in one asymmetric unit (slate) interacts with another hNaa60 molecule in a neighboring asymmetric unit (cyan). A close view of the interaction is shown in red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule participating in the interaction are shown as yellow and green sticks, respectively. (B) The α5 helix of hNaa60(1-199) in one asymmetric unit (yellow) interacts with another hNaa60 molecule in the neighboring asymmetric units (green). A close view of the interaction is shown in the red box. Side-chains of hydrophobic residues on α5 helix and the neighboring molecule (green) participating in the interaction are shown as yellow and green sticks, respectively. The third molecule (pink) does not directly interact with the α5 helix. (C) Superposition of hNaa60(1-199) (yellow) and hNaa60(1-242) (cyan) showing conformational change of the β7-β8 hairpin in these two structures. (D,E) Superposition of Hat1p/H4 (gray, drawn from PDB 4PSW) with hNaa60(1-242) (cyan, D) or hNaa60(1-199) (yellow, E). The histone H4 peptide (a KAT substrate) bound to Hat1p is shown in purple (D,E), while the peptide bound to hNaa50 (a NAT substrate, drawn from PDB 3TFY) is shown in orange (Nt-peptide) after superimposing hNaa50 (not shown in figure) on hNaa60 (D). The α-amine of the NAT substrate and ε-amine of the KAT substrate (along with the lysine side-chain) subject to acetylation are shown as sticks.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44987</offset><text>Electron density map of the active site.</text></passage><passage><infon key="file">srep31425-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45028</offset><text>The 2Fo-Fc maps contoured at 1.0σ are shown for hNaa60(1-242)/Ac-CoA (A), hNaa60(1-199)/CoA (B) and hNaa60(1-199) F34A/CoA (C). The putative substrate peptide binding site is indicated by the peptide (shown as pink sticks) from the hNaa50/CoA/peptide complex structure after superimposing hNaa50 on the hNaa60 structures determined in this study. The black arrow indicates the α-amine of the first Met (M1) (all panels). The purple arrow indicates the acetyl moiety of Ac-CoA (A). The red arrow indicates the alternative conformation of the thiol moiety of the co-enzyme when Phe 34 side-chain is displaced (B) or mutated to Ala (C).</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>45670</offset><text>Structural basis for hNaa60 catalytic activity.</text></passage><passage><infon key="file">srep31425-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45718</offset><text>(A) Superposition of hNaa60 active site (cyan) on that of hNaa50 (pink, PDB 3TFY). Side-chains of key catalytic and substrate-binding residues are highlighted as sticks. The malonate molecule in the hNaa60(1-242)/Ac-CoA structure and the peptide in the hNaa50/CoA/peptide structure are shown as purple and yellow sticks respectively. (B) A close view of the active site of hNaa60. Residues Glu 37, Tyr 97 and His 138 in hNaa60 (cyan) and corresponding residues (Tyr 73 and His 112) in hNaa50 (pink) as well as the side-chain of corresponding residues (Glu 24, His 72 and His 111) in complexed formed hNaa10p (warmpink) are highlighted as sticks. The water molecules participating in catalysis in the hNaa60 and hNaa50 structures are showed as green and red spheres, separately. (C) The interaction between the malonate molecule and surrounding residues observed in the hNaa60(1-242)/Ac-CoA structure. The yellow dotted lines indicate the hydrogen bonds. (D) A zoomed view of β3-β4 loop of hNaa60. Key residues discussed in the text (cyan), the malonate (purple) and Ac-CoA (gray) are shown as sticks. The yellow dotted lines indicate the salt bridges.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>46876</offset><text>Catalytic activity of hNaa60 and mutant proteins.</text></passage><passage><infon key="file">srep31425-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>46926</offset><text>(A) Catalytic efficiency (shown as kcat/Km values) of hNaa60 (1-199) WT and mutants. Error bars indicate the Standard Error (SE). (B) CD spectra of wild-type and mutant proteins from 250 nm to 190 nm. The sample concentration was 4.5 μM in 20 mM Tris, pH 8.0, 150 mM NaCl, 1% glycerol and 1 mM TCEP at room temperature.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>47259</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50">Structure and PDB ID</th><th align="center" valign="top" charoff="50">hNaa60(1-242)/Ac-CoA 5HGZ</th><th align="center" valign="top" charoff="50">hNaa60(1-199)/CoA 5HH0</th><th align="center" valign="top" charoff="50">hNaa60(1-199)F34A/CoA 5HH1</th></tr></thead><tbody valign="top"><tr><td colspan="4" align="left" valign="top" charoff="50">Data collection<xref ref-type="fn" rid="t1-fn1">*</xref></td></tr><tr><td align="left" valign="top" charoff="50"> Space group</td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub></td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic></td><td align="center" valign="top" charoff="50"><italic>P2</italic><sub><italic>1</italic></sub><italic>2</italic><sub><italic>1</italic></sub><italic>2</italic></td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>a, b, c</italic> (Å)</td><td align="center" valign="top" charoff="50">53.3, 57.4, 68.8</td><td align="center" valign="top" charoff="50">67.8, 73.8, 43.2</td><td align="center" valign="top" charoff="50">66.7, 74.0, 43.5</td></tr><tr><td align="left" valign="top" charoff="50"> α,β,γ (°)</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td><td align="center" valign="top" charoff="50">90.0, 90.0, 90.0</td></tr><tr><td align="left" valign="top" charoff="50">Resolution (Å)</td><td align="center" valign="top" charoff="50">50–1.38 (1.42–1.38)</td><td align="center" valign="top" charoff="50">50–1.60 (1.66–1.60)</td><td align="center" valign="top" charoff="50">50–1.80 (1.86–1.80)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic><sub>p.i.m.</sub>(%)<xref ref-type="fn" rid="t1-fn2">**</xref></td><td align="center" valign="top" charoff="50">3.0 (34.4)</td><td align="center" valign="top" charoff="50">2.1 (32.5)</td><td align="center" valign="top" charoff="50">2.6 (47.8)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>I</italic>/<italic>σ</italic></td><td align="center" valign="top" charoff="50">21.5 (2.0)</td><td align="center" valign="top" charoff="50">31.8 (2.0)</td><td align="center" valign="top" charoff="50">28.0 (2.4)</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">99.8 (99.1)</td><td align="center" valign="top" charoff="50">99.6 (98.5)</td><td align="center" valign="top" charoff="50">99.9 (99.7)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">6.9 (5.0)</td><td align="center" valign="top" charoff="50">6.9 (6.2)</td><td align="center" valign="top" charoff="50">6.3 (5.9)</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Refinement</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">25.81–1.38</td><td align="center" valign="top" charoff="50">33.55–1.60</td><td align="center" valign="top" charoff="50">43.52–1.80</td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections</td><td align="center" valign="top" charoff="50">43660</td><td align="center" valign="top" charoff="50">28588</td><td align="center" valign="top" charoff="50">20490</td></tr><tr><td align="left" valign="top" charoff="50"><italic> R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">0.182/0.192</td><td align="center" valign="top" charoff="50">0.181/0.184</td><td align="center" valign="top" charoff="50">0.189/0.209</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">No. atoms</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">1717</td><td align="center" valign="top" charoff="50">1576</td><td align="center" valign="top" charoff="50">1566</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">116</td><td align="center" valign="top" charoff="50">96</td><td align="center" valign="top" charoff="50">96</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">289</td><td align="center" valign="top" charoff="50">258</td><td align="center" valign="top" charoff="50">168</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50"><italic>B</italic>-factors</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">23.8</td><td align="center" valign="top" charoff="50">32.0</td><td align="center" valign="top" charoff="50">37.4</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">22.2</td><td align="center" valign="top" charoff="50">34.6</td><td align="center" valign="top" charoff="50">43.7</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">35.1</td><td align="center" valign="top" charoff="50">46.4</td><td align="center" valign="top" charoff="50">49.1</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">R.m.s. deviations</td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.018</td><td align="center" valign="top" charoff="50">0.017</td><td align="center" valign="top" charoff="50">0.015</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="center" valign="top" charoff="50">1.529</td><td align="center" valign="top" charoff="50">1.651</td><td align="center" valign="top" charoff="50">1.581</td></tr><tr><td colspan="4" align="left" valign="top" charoff="50">Ramachandran Plot</td></tr><tr><td align="left" valign="top" charoff="50"> Favoured region</td><td align="center" valign="top" charoff="50">98.98%</td><td align="center" valign="top" charoff="50">98.93%</td><td align="center" valign="top" charoff="50">98.96%</td></tr><tr><td align="left" valign="top" charoff="50"> Allowed region</td><td align="center" valign="top" charoff="50">1.02%</td><td align="center" valign="top" charoff="50">1.07%</td><td align="center" valign="top" charoff="50">1.04%</td></tr><tr><td align="left" valign="top" charoff="50"> Outliers</td><td align="center" valign="top" charoff="50">0.00%</td><td align="center" valign="top" charoff="50">0.00%</td><td align="center" valign="top" charoff="50">0.00%</td></tr></tbody></table>
</infon><offset>47302</offset><text>Structure and PDB ID hNaa60(1-242)/Ac-CoA 5HGZ hNaa60(1-199)/CoA 5HH0 hNaa60(1-199)F34A/CoA 5HH1 Data collection* Space group P212121 P21212 P21212 Cell dimensions a, b, c (Å) 53.3, 57.4, 68.8 67.8, 73.8, 43.2 66.7, 74.0, 43.5 α,β,γ (°) 90.0, 90.0, 90.0 90.0, 90.0, 90.0 90.0, 90.0, 90.0 Resolution (Å) 50–1.38 (1.42–1.38) 50–1.60 (1.66–1.60) 50–1.80 (1.86–1.80) Rp.i.m.(%)** 3.0 (34.4) 2.1 (32.5) 2.6 (47.8) I/σ 21.5 (2.0) 31.8 (2.0) 28.0 (2.4) Completeness (%) 99.8 (99.1) 99.6 (98.5) 99.9 (99.7) Redundancy 6.9 (5.0) 6.9 (6.2) 6.3 (5.9) Refinement Resolution (Å) 25.81–1.38 33.55–1.60 43.52–1.80 No. reflections 43660 28588 20490 Rwork/Rfree 0.182/0.192 0.181/0.184 0.189/0.209 No. atoms Protein 1717 1576 1566 Ligand/ion 116 96 96 Water 289 258 168 B-factors Protein 23.8 32.0 37.4 Ligand/ion 22.2 34.6 43.7 Water 35.1 46.4 49.1 R.m.s. deviations Bond lengths (Å) 0.018 0.017 0.015 Bond angles (°) 1.529 1.651 1.581 Ramachandran Plot Favoured region 98.98% 98.93% 98.96% Allowed region 1.02% 1.07% 1.04% Outliers 0.00% 0.00% 0.00% </text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>48477</offset><text>*Values in parentheses are for highest-resolution shell. One crystal was used for each data set.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>48574</offset><text>**Rp.i.m., a redundancy-independent R factor was used to evaluate the diffraction data quality as was proposed by Evans.</text></passage></document></collection>
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<collection><source>PMC</source><date>20230811</date><key>pmc.key</key><document><id>5012862</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.7554/eLife.18972</infon><infon key="article-id_pmc">5012862</infon><infon key="article-id_pmid">27529188</infon><infon key="article-id_publisher-id">18972</infon><infon key="elocation-id">e18972</infon><infon key="kwd">encapsulin encapsulated ferritin ferritin Rhodospirillum rubrum Other</infon><infon key="license">This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.</infon><infon key="name_0">surname:He;given-names:Didi</infon><infon key="name_1">surname:Hughes;given-names:Sam</infon><infon key="name_10">surname:Losick;given-names:Richard</infon><infon key="name_11">surname:He;given-names:Didi</infon><infon key="name_12">surname:Hughes;given-names:Sam</infon><infon key="name_13">surname:Altenbach;given-names:Kirsten</infon><infon key="name_14">surname:Clarke;given-names:David J</infon><infon key="name_15">surname:Tarrant;given-names:Emma</infon><infon key="name_16">surname:Waldron;given-names:Kevin J</infon><infon key="name_17">surname:Clarke;given-names:David J</infon><infon key="name_18">surname:Marles-Wright;given-names:Jon</infon><infon key="name_19">surname:Marles-Wright;given-names:Jon</infon><infon key="name_2">surname:Vanden-Hehir;given-names:Sally</infon><infon key="name_3">surname:Georgiev;given-names:Atanas</infon><infon key="name_4">surname:Altenbach;given-names:Kirsten</infon><infon key="name_5">surname:Tarrant;given-names:Emma</infon><infon key="name_6">surname:Mackay;given-names:C Logan</infon><infon key="name_7">surname:Waldron;given-names:Kevin J</infon><infon key="name_8">surname:Clarke;given-names:David J</infon><infon key="name_9">surname:Marles-Wright;given-names:Jon</infon><infon key="section_type">TITLE</infon><infon key="title">Author Keywords Research Organism</infon><infon key="type">front</infon><infon key="volume">5</infon><infon key="year">2016</infon><offset>0</offset><text>Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>118</offset><text>Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1107</offset><text>DOI: http://dx.doi.org/10.7554/eLife.18972.001</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1154</offset><text>eLife digest</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1167</offset><text>Iron is essential for life as it is a key component of many different enzymes that participate in processes such as energy production and metabolism. However, iron can also be highly toxic to cells because it readily reacts with oxygen. This reaction can damage DNA, proteins and the membranes that surround cells.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1482</offset><text>To balance the cell’s need for iron against its potential damaging effects, organisms have evolved iron storage proteins known as ferritins that form cage-like structures. The ferritins convert iron into a less reactive form that is mineralised and safely stored in the central cavity of the ferritin cage and is available for cells when they need it.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1836</offset><text>Recently, a new family of ferritins known as encapsulated ferritins have been found in some microorganisms. These ferritins are found in bacterial genomes with a gene that codes for a protein cage called an encapsulin. Although the structure of the encapsulin cage is known to look like the shell of a virus, the structure that the encapsulated ferritin itself forms is not known. It is also not clear how encapsulin and the encapsulated ferritin work together to store iron.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2312</offset><text>He et al. have now used the techniques of X-ray crystallography and mass spectrometry to determine the structure of the encapsulated ferritin found in some bacteria. The encapsulated ferritin forms a ring-shaped doughnut in which ten subunits of ferritin are arranged in a ring; this is totally different from the enclosed cages that other ferritins form.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2668</offset><text>Biochemical studies revealed that the encapsulated ferritin is able to convert iron into a less reactive form, but it cannot store iron on its own since it does not form a cage. Thus, the encapsulated ferritin needs to be housed within the encapsulin cage to store iron.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>2939</offset><text>Further work is needed to investigate how iron moves into the encapsulin cage to reach the ferritin proteins. Some organisms have both standard ferritin cages and encapsulated ferritins; why this is the case also remains to be discovered.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>3178</offset><text>DOI: http://dx.doi.org/10.7554/eLife.18972.002</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>3225</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3238</offset><text>Encapsulin nanocompartments are a family of proteinaceous metabolic compartments that are widely distributed in bacteria and archaea. They share a common architecture, comprising an icosahedral shell formed by the oligomeric assembly of a protein, encapsulin, that is structurally related to the HK97 bacteriophage capsid protein gp5. Gp5 is known to assemble as a 66 nm diameter icosahedral shell of 420 subunits. In contrast, both the Pyrococcus furiosus and Myxococcus xanthus encapsulin shell-proteins form 32 nm icosahedra with 180 subunits; while the Thermotoga maritima encapsulin is smaller still with a 25 nm, 60-subunit icosahedron. The high structural similarity of the encapsulin shell-proteins to gp5 suggests a common evolutionary origin for these proteins.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4011</offset><text>The genes encoding encapsulin proteins are found downstream of genes for dye-dependent peroxidase (DyP) family enzymes, or encapsulin-associated ferritins (EncFtn). Enzymes in the DyP family are active against polyphenolic compounds such as azo dyes and lignin breakdown products; although their physiological function and natural substrates are not known. Ferritin family proteins are found in all kingdoms and have a wide range of activities, including ribonucleotide reductase, protecting DNA from oxidative damage, and iron storage. The classical iron storage ferritin nanocages are found in all kingdoms and are essential in eukaryotes; they play a central role in iron homeostasis, where they protect the cell from toxic free Fe2+ by oxidizing it and storing the resulting Fe3+ as ferrihydrite minerals within their central cavity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4849</offset><text>The encapsulin-associated enzymes are sequestered within the icosahedral shell through interactions between the shell’s inner surface and a short localization sequence (Gly-Ser-Leu-Lys) appended to their C-termini. This motif is well-conserved, and the addition of this sequence to heterologous proteins is sufficient to direct them to the interior of encapsulins.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5217</offset><text>A recent study of the Myxococcus xanthus encapsulin showed that it sequesters a number of different EncFtn proteins and acts as an ‘iron-megastore’ to protect these bacteria from oxidative stress. At 32 nm in diameter, it is much larger than other members of the ferritin superfamily, such as the 12 nm 24-subunit classical ferritin nanocage and the 8 nm 12-subunit Dps (DNA-binding protein from starved cells) complex; and is thus capable of sequestering up to ten times more iron than these ferritins. The primary sequences of EncFtn proteins have Glu-X-X-His metal coordination sites, which are shared features of the ferritin family proteins. Secondary structure prediction identifies two major α-helical regions in these proteins; this is in contrast to other members of the ferritin superfamily, which have four major α-helices (Supplementary file 1). The ‘half-ferritin’ primary sequence of the EncFtn family and their association with encapsulin nanocompartments suggests a distinct biochemical and structural organization to other ferritin family proteins. The Rhodospirillum rubrum EncFtn protein (Rru_A0973) shares 33% protein sequence identity with the M. xanthus (MXAN_4464), 53% with the T. maritima (Tmari_0787), and 29% with the P. furiosus (PF1192) homologues. The GXXH motifs are strictly conserved in each of these species (Supplementary file 1).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6594</offset><text>Here we investigate the structure and biochemistry of EncFtn in order to understand iron storage within the encapsulin nanocompartment. We have produced recombinant encapsulin (Enc) and EncFtn from the aquatic purple-sulfur bacterium R. rubrum, which serves as a model organism for the study of the control of the bacterial nitrogen fixation machinery, in Escherichia coli. Analysis by transmission electron microscopy (TEM) indicates that their co-expression leads to the production of an icosahedral nanocompartment with encapsulated EncFtn. The crystal structure of a truncated hexahistidine-tagged variant of the EncFtn protein (EncFtnsH) shows that it forms a decameric structure with an annular ‘ring-doughnut’ topology, which is distinct from the four-helical bundles of the 24meric ferritins and dodecahedral DPS proteins. We identify a symmetrical iron bound ferroxidase center (FOC) formed between subunits in the decamer and additional metal-binding sites close to the center of the ring and on the outer surface. We also demonstrate the metal-dependent assembly of EncFtn decamers using native PAGE, analytical gel-filtration, and native mass spectrometry. Biochemical assays show that EncFtn is active as a ferroxidase enzyme. Through site-directed mutagenesis we show that the conserved glutamic acid and histidine residues in the FOC influence protein assembly and activity. We use our combined structural and biochemical data to propose a model for the EncFtn-catalyzed sequestration of iron within the encapsulin shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8136</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8144</offset><text>Assembly of R. rubrum EncFtn encapsulin nanocompartments in E. coli</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8212</offset><text>Full-frame transmission electron micrographs of R. rubrum nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8288</offset><text>(A/B) Negative stain TEM image of recombinant R. rubrum encapsulin and EncFtn-Enc nanocompartments. All samples were imaged at 143,000 x magnification; the scale bar length corresponds to 50 nm. (C) Histogram showing the distribution of nanocompartment diameters. A model Gaussian nonlinear least square function was fitted to the data to obtain a mean diameter of 24.6 nm with a standard deviation of 2.0 nm for encapsulin (grey) and a mean value of 23.9 nm with a standard deviation of 2.2 nm for co-expressed EncFtn and encapsulin (EncFtn-Enc, black).</text></passage><passage><infon key="file">elife-18972-fig1-figsupp1.jpg</infon><infon key="id">fig1s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8845</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.004</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>8892</offset><text>Purification of recombinant R. rubrum encapsulin nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>8959</offset><text>(A) Recombinantly expressed encapsulin (Enc) and co-expressed EncFtn-Enc were purified by sucrose gradient ultracentrifugation from E. coli B834(DE3) grown in SeMet medium. Samples were resolved by 18% acrylamide SDS-PAGE; the position of the proteins found in the complexes as resolved on the gel are shown with arrows. (B/C) Negative stain TEM image of recombinant encapsulin and EncFtn-Enc nanocompartments. Samples were imaged at 143,000 x magnification, with scale bar shown as 25 nm. Representative encapsulin and EncFtn-Enc complexes are indicated with red arrows.</text></passage><passage><infon key="file">elife-18972-fig1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>9531</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.003</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9578</offset><text>We produced recombinant R. rubrum encapsulin nanocompartments in E. coli by co-expression of the encapsulin (Rru_A0974) and EncFtn (Rru_A0973) proteins, and purified these by sucrose gradient ultra-centrifugation (Figure 1A). TEM imaging of uranyl acetate-stained samples revealed that, when expressed in isolation, the encapsulin protein forms empty compartments with an average diameter of 24 nm (Figure 1B and Figure 1—figure supplement 1A/C), consistent with the appearance and size of the T. maritima encapsulin. We were not able to resolve any higher-order structures of EncFtn by TEM. Protein purified from co-expression of the encapsulin and EncFtn resulted in 24 nm compartments with regions in the center that exclude stain, consistent with the presence of the EncFtn within the encapsulin shell (Figure 1C and Figure 1—figure supplement 1B/C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10437</offset><text>R. rubrum EncFtn forms a metal-ion stabilized decamer in solution</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>10504</offset><text>Purification of recombinant R. rubrum EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>10552</offset><text>(A) Recombinant SeMet-labeled EncFtnsH produced with 1 mM Fe(NH4)2(SO4)2 in the growth medium was purified by nickel affinity chromatography and size-exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare). Chromatogram traces measured at 280 nm and 315 nm are shown with the results from ICP-MS analysis of the iron content of the fractions collected during the experiment. The peak around 73 ml corresponds to a molecular weight of around 130 kDa when compared to calibration standards; this is consistent with a decamer of EncFtnsH. The small peak at 85 ml corresponds to the 13 kDa monomer compared to the standards. Only the decamer peak contains significant amounts of iron as indicated by the ICP-MS analysis. (B) Peak fractions from the gel filtration run were resolved by 15% acrylamide SDS-PAGE and stained with Coomassie blue stain. The bands around 13 kDa and 26 kDa correspond to EncFtnsH, as identified by MALDI peptide mass fingerprinting. The band at 13 kDa is consistent with the monomer mass, while the band at 26 kDa is consistent with a dimer of EncFtnsH. The dimer species only appears in the decamer fractions. (C) SEC-MALLS analysis of EncFtnsH from decamer fractions and monomer fractions allows assignment of an average mass of 132 kDa to decamer fractions and 13 kDa to monomer fractions, consistent with decamer and monomer species (Table 2).</text></passage><passage><infon key="file">elife-18972-fig2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11944</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.005</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>11991</offset><text>Determination of the Fe/EncFtnsH protein ratio by ICP-MS. EncFtnsH was purified as a SeMet derivative from E. coli B834(DE3) cells grown in SeMet medium with 1 mM Fe(NH4)2(SO4)2. Fractions from SEC were collected, acidified and analysed by ICP-MS. EncFtnsH concentration was calculated based on the presence of two SeMet per mature monomer. Samples where the element was undetectable are labelled with n.d. These data were collected from EncFtnsH fractions from a single gel-filtration run.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>12482</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.006</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="2" colspan="1">Peak</th><th rowspan="2" colspan="1">EncFtn<sub>sH</sub>
<break/>retention volume (ml)</th><th colspan="4" rowspan="1">Element concentration (µM)</th><th rowspan="2" colspan="1">Derived EncFtn<sub>sH</sub>concentration (µM)</th><th rowspan="2" colspan="1">Derived Fe/ <break/>EncFtn<sub>sH</sub> monomer</th></tr><tr><th rowspan="1" colspan="1">Ca</th><th rowspan="1" colspan="1">Fe</th><th rowspan="1" colspan="1">Zn</th><th rowspan="1" colspan="1">Se</th></tr></thead><tbody><tr><td rowspan="8" colspan="1">Decamer</td><td rowspan="1" colspan="1">66.5</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">6.7</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">24.6</td><td rowspan="1" colspan="1"><p>12.3</p></td><td rowspan="1" colspan="1"><p>0.5</p></td></tr><tr><td rowspan="1" colspan="1">68.3</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">28.4</td><td rowspan="1" colspan="1">n.d</td><td rowspan="1" colspan="1">124.5</td><td rowspan="1" colspan="1"><p>62.3</p></td><td rowspan="1" colspan="1"><p>0.5</p></td></tr><tr><td rowspan="1" colspan="1">70.1</td><td rowspan="1" colspan="1">2.9</td><td rowspan="1" colspan="1">93.7</td><td rowspan="1" colspan="1">2.4</td><td rowspan="1" colspan="1">301.7</td><td rowspan="1" colspan="1"><p>150.9</p></td><td rowspan="1" colspan="1"><p>0.6</p></td></tr><tr><td rowspan="1" colspan="1">71.9</td><td rowspan="1" colspan="1">6.9</td><td rowspan="1" colspan="1">120.6</td><td rowspan="1" colspan="1">3.7</td><td rowspan="1" colspan="1">379.8</td><td rowspan="1" colspan="1"><p>189.9</p></td><td rowspan="1" colspan="1"><p>0.6</p></td></tr><tr><td rowspan="1" colspan="1">73.7</td><td rowspan="1" colspan="1">1.9</td><td rowspan="1" colspan="1">64.4</td><td rowspan="1" colspan="1">0.8</td><td rowspan="1" colspan="1">240.6</td><td rowspan="1" colspan="1"><p>120.3</p></td><td rowspan="1" colspan="1"><p>0.5</p></td></tr><tr><td rowspan="1" colspan="1">75.5</td><td rowspan="1" colspan="1">0.9</td><td rowspan="1" colspan="1">21.1</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">101.7</td><td rowspan="1" colspan="1"><p>50.8</p></td><td rowspan="1" colspan="1"><p>0.4</p></td></tr><tr><td rowspan="1" colspan="1">77.3</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">6.2</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">42.6</td><td rowspan="1" colspan="1"><p>21.3</p></td><td rowspan="1" colspan="1"><p>0.3</p></td></tr><tr><td rowspan="1" colspan="1">79.1</td><td rowspan="1" colspan="1">0.1</td><td rowspan="1" colspan="1">2.4</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">26.5</td><td rowspan="1" colspan="1"><p>13.3</p></td><td rowspan="1" colspan="1"><p>0.2</p></td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">80.9</td><td rowspan="1" colspan="1">1.0</td><td rowspan="1" colspan="1">1.5</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">22.3</td><td rowspan="1" colspan="1"><p>11.2</p></td><td rowspan="1" colspan="1"><p>0.1</p></td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1">82.7</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">0.2</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">29.2</td><td rowspan="1" colspan="1"><p>14.6</p></td><td rowspan="1" colspan="1"><p>n.d</p></td></tr><tr><td rowspan="5" colspan="1">Monomer</td><td rowspan="1" colspan="1">84.5</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">0.1</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">34.9</td><td rowspan="1" colspan="1"><p>17.5</p></td><td rowspan="1" colspan="1"><p>n.d</p></td></tr><tr><td rowspan="1" colspan="1">86.3</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">28.9</td><td rowspan="1" colspan="1"><p>14.4</p></td><td rowspan="1" colspan="1"><p>n.d</p></td></tr><tr><td rowspan="1" colspan="1">88.1</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">17.4</td><td rowspan="1" colspan="1"><p>8.7</p></td><td rowspan="1" colspan="1"><p>n.d.</p></td></tr><tr><td rowspan="1" colspan="1">89.9</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">5.5</td><td rowspan="1" colspan="1"><p>2.8</p></td><td rowspan="1" colspan="1"><p>n.d.</p></td></tr><tr><td rowspan="1" colspan="1">91.7</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">n.d.</td><td rowspan="1" colspan="1">0.1</td><td rowspan="1" colspan="1"><p>0.07</p></td><td rowspan="1" colspan="1"><p>0.2</p></td></tr></tbody></table>
</infon><offset>12529</offset><text>Peak EncFtnsHretention volume (ml) Element concentration (µM) Derived EncFtnsHconcentration (µM) Derived Fe/ EncFtnsH monomer Ca Fe Zn Se Decamer 66.5 n.d. 6.7 n.d. 24.6 12.3 0.5 68.3 n.d. 28.4 n.d 124.5 62.3 0.5 70.1 2.9 93.7 2.4 301.7 150.9 0.6 71.9 6.9 120.6 3.7 379.8 189.9 0.6 73.7 1.9 64.4 0.8 240.6 120.3 0.5 75.5 0.9 21.1 n.d. 101.7 50.8 0.4 77.3 n.d. 6.2 n.d. 42.6 21.3 0.3 79.1 0.1 2.4 n.d. 26.5 13.3 0.2 80.9 1.0 1.5 n.d. 22.3 11.2 0.1 82.7 n.d. 0.2 n.d. 29.2 14.6 n.d Monomer 84.5 n.d. 0.1 n.d. 34.9 17.5 n.d 86.3 n.d. n.d n.d. 28.9 14.4 n.d 88.1 n.d. n.d. n.d. 17.4 8.7 n.d. 89.9 n.d. n.d. n.d. 5.5 2.8 n.d. 91.7 n.d. n.d. n.d. 0.1 0.07 0.2 </text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13228</offset><text>Estimates of EncFtnsH molecular weight from SEC-MALLS analysis. EncFtnsH was purified from E. coli BL21(DE3) grown in minimal medium (MM) by nickel affinity chromatography and size-exclusion chromatography. Fractions from two peaks (decamer and monomer) were pooled separately (Figure 1C) and analysed by SEC-MALLS using a Superdex 200 10/300 GL column (GE Healthcare) and Viscotek SEC-MALLS instruments (Malvern Instruments) (Figure 2C). The decamer and monomer peaks were both symmetric and monodisperse, allowing the estimation of the molecular weight of the species in these fractions. The molecular weights are quoted to the nearest kDa due to the resolution limit of the instrument. The proteins analyzed by SEC-MALLS came from single protein preparation.</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>13997</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.007</text></passage><passage><infon key="file">tbl2.xml</infon><infon key="id">tbl2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Molecular Weight (kDa)</th><th rowspan="1" colspan="1">Decamer peak</th><th rowspan="1" colspan="1">Monomer peak</th></tr></thead><tbody><tr><td rowspan="1" colspan="1">Theoretical</td><td rowspan="1" colspan="1">133</td><td rowspan="1" colspan="1">13</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-decamer fractions</td><td rowspan="1" colspan="1">132</td><td rowspan="1" colspan="1">15</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">126</td><td rowspan="1" colspan="1">13</td></tr></tbody></table>
</infon><offset>14044</offset><text>Molecular Weight (kDa) Decamer peak Monomer peak Theoretical 133 13 EncFtnsH-decamer fractions 132 15 EncFtnsH-monomer fractions 126 13 </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14189</offset><text>We purified recombinant R. rubrum EncFtn as both the full-length sequence (140 amino acids) and a truncated C-terminal hexahistidine-tagged variant (amino acids 1–96 plus the tag; herein EncFtnsH). In both cases the elution profile from size-exclusion chromatography (SEC) displayed two peaks (Figure 2A). SDS-PAGE analysis of fractions from these peaks showed that the high molecular weight peak was partially resistant to SDS and heat-induced denaturation; in contrast, the low molecular weight peak was consistent with monomeric mass of 13 kDa (Figure 2B). MALDI peptide mass fingerprinting of these bands confirmed the identity of both as EncFtn. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of the SEC fractions showed 100 times more iron in the oligomeric fraction than the monomer (Figure 2A, blue scatter points; Table 1), suggesting that EncFtn oligomerization is associated with iron binding. In order to determine the iron-loading stoichiometry in the EncFtn complex, further ICP-MS experiments were performed using selenomethionine (SeMet)-labelled protein EncFtn (Table 1). In these experiments, we observed sub-stoichiometric metal binding, which is in contrast to the classical ferritins. Size-exclusion chromatography with multi-angle laser light scattering (SEC-MALLS) analysis of samples taken from each peak gave calculated molecular weights consistent with a decamer for the high molecular weight peak and a monomer for the low molecular weight peak (Figure 2C, Table 2).</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>15702</offset><text>Effect of metal ions on the oligomeric state of EncFtnsH in solution.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>15773</offset><text>(A/B) EncFtnsH-monomer was incubated with one mole equivalent of various metal salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column. Co2+ and Zn2+ induced the formation of the decameric form of EncFtnsH; while Mn2+, Mg2+ and Fe3+ did not significantly alter the oligomeric state of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp1.jpg</infon><infon key="id">fig3s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16105</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.009</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16152</offset><text>PAGE analysis of the effect of metal ions on the oligomeric state of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16231</offset><text>50 µM EncFtnsH monomer or decamer samples were mixed with equal molar metal ions including Fe2+, Co2+, Zn2+, Mn2+, Ca2+, Mg2+ and Fe3+, which were analyzed by Native PAGE alongside SDS-PAGE. (A) 10% Native PAGE analysis of EncFtnsH monomer fractions mixed with various metal solutions; (B) 10% Native PAGE analysis of EncFtnsH decamer fractions mixed with various metal solutions; (C) 15% SDS-PAGE analysis on the mixtures of EncFtnsH monomer fractions and metal solutions; (D) 15% SDS-PAGE analysis on the mixtures of EncFtnsH decamer fractions and metal solutions.</text></passage><passage><infon key="file">elife-18972-fig3-figsupp2.jpg</infon><infon key="id">fig3s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16802</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.010</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16849</offset><text>Effect of Fe2+ and protein concentration on the oligomeric state of EncFtnsH in solution.</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16939</offset><text>(A) Recombinant EncFtnsH was purified by Gel filtration Superdex 200 chromatography from E. coli BL21(DE3) grown in MM or in MM supplemented with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+). A higher proportion of decamer (peak between 65 and 75 ml) is seen in the sample purified from MM+Fe2+ compared to EncFtnsH-MM, indicating that Fe2+ facilitates the multimerization of EncFtnsH
in vivo. (B) EncFtnsH-monomer was incubated with one molar equivalent of Fe2+ salts for two hours prior to analytical gel-filtration using a Superdex 200 PC 3.2/30 column (GE Healthcare). Both Fe2+ salts tested induced the formation of decamer indicated by the peak between 1.2 and 1.6 ml. Monomeric and decameric samples of EncFtnsH are shown as controls. Peaks around 0.8 ml were seen as protein aggregation. (C) Analytical gel filtration of EncFtn monomer at different concentrations to illustrate the effect of protein concentration on multimerization. The major peak shows a shift towards a dimer species at high concentration of protein, but the ratio of this peak (1.5–1.8 ml) to the decamer peak (1.2–1.5 ml) does not change when compared to the low concentration sample.</text></passage><passage><infon key="file">elife-18972-fig3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>18097</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.008</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>18144</offset><text>Gel-filtration peak area ratios for EncFtnsH decamer and monomer on addition of different metal ions. EncFtnsH was produced in E. coli BL21(DE3) cultured in MM and MM with 1 mM Fe(NH4)2(SO4)2 (MM+Fe2+) and purified by gel-filtration chromatography using an Superdex 200 16/60 column (GE Healthcare). Monomer fractions of EncFtnsH purified from MM were pooled and run in subsequent analytical gel-filtration runs over the course of three days. Samples of EncFtnsH monomer were incubated with one molar equivalent of metal ion salts at room temperature for two hours before analysis by analytical gel filtration chromatography (AGF) using a Superdex 200 10/300 GL column. The area for resulting protein peaks were calculated using the Unicorn software (GE Healthcare); peak ratios were calculated to quantify the propensity of EncFtnsH to multimerize in the presence of the different metal ions. The change in the ratios of monomer to decamer over the three days of experiments may be a consequence of experimental variability, or the propensity of this protein to equilibrate towards decamer over time. The increased decamer: monomer ratio seen in the presence of Fe2+, Co2+, and Zn2+ indicates that these metal ions facilitate multimerization of the EncFtnsH protein, while the other metal ions tested do not appear to induce multimerization. The analytical gel filtration experiment was repeated twice using two independent preparations of protein, of which values calculated from one sample are presented here.</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>19662</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.011</text></passage><passage><infon key="file">tbl3.xml</infon><infon key="id">tbl3</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Method</th><th rowspan="1" colspan="1">Sample</th><th rowspan="1" colspan="1">Monomer area</th><th rowspan="1" colspan="1">Decamer area</th><th rowspan="1" colspan="1">Decamer/Monomer</th></tr></thead><tbody><tr><td rowspan="2" colspan="1">Gel filtration Superdex 200 chromatography</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-MM</td><td rowspan="1" colspan="1">64.3</td><td rowspan="1" colspan="1">583.6</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-MM+Fe<sup>2+</sup></td><td rowspan="1" colspan="1">1938.4</td><td rowspan="1" colspan="1">426.4</td><td rowspan="1" colspan="1">4.5</td></tr><tr><td rowspan="4" colspan="1">Analytical Gel filtration Day1</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-decamer fractions</td><td rowspan="1" colspan="1">20.2</td><td rowspan="1" colspan="1">1.8</td><td rowspan="1" colspan="1">11.2</td></tr><tr><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">2.9</td><td rowspan="1" colspan="1">21.9</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">Fe(NH<sub>4)2</sub>(SO<sub>4)2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">11.0</td><td rowspan="1" colspan="1">13.0</td><td rowspan="1" colspan="1">0.8</td></tr><tr><td rowspan="1" colspan="1">FeSO<sub>4</sub>-HCl/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">11.3</td><td rowspan="1" colspan="1">11.4</td><td rowspan="1" colspan="1">1.0</td></tr><tr><td rowspan="5" colspan="1">Analytical Gel filtration Day2</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">8.3</td><td rowspan="1" colspan="1">22.8</td><td rowspan="1" colspan="1">0.4</td></tr><tr><td rowspan="1" colspan="1">CoCl<sub>2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">17.7</td><td rowspan="1" colspan="1">14.5</td><td rowspan="1" colspan="1">1.2</td></tr><tr><td rowspan="1" colspan="1">MnCl<sub>2</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">3.1</td><td rowspan="1" colspan="1">30.5</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="1" colspan="1">ZnSO<sub>4</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">20.4</td><td rowspan="1" colspan="1">9.0</td><td rowspan="1" colspan="1">2.3</td></tr><tr><td rowspan="1" colspan="1">FeCl<sub>3</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">3.9</td><td rowspan="1" colspan="1">28.6</td><td rowspan="1" colspan="1">0.1</td></tr><tr><td rowspan="3" colspan="1">Analytical Gel filtration Day3</td><td rowspan="1" colspan="1">EncFtn<sub>sH</sub>-monomer fractions</td><td rowspan="1" colspan="1">6.3</td><td rowspan="1" colspan="1">23.4</td><td rowspan="1" colspan="1">0.3</td></tr><tr><td rowspan="1" colspan="1">MgSO<sub>4</sub>/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">5.8</td><td rowspan="1" colspan="1">30.2</td><td rowspan="1" colspan="1">0.2</td></tr><tr><td rowspan="1" colspan="1">Ca acetate/EncFtn<sub>sH</sub>-monomer</td><td rowspan="1" colspan="1">5.6</td><td rowspan="1" colspan="1">25.2</td><td rowspan="1" colspan="1">0.2</td></tr></tbody></table>
</infon><offset>19709</offset><text>Method Sample Monomer area Decamer area Decamer/Monomer Gel filtration Superdex 200 chromatography EncFtnsH-MM 64.3 583.6 0.1 EncFtnsH-MM+Fe2+ 1938.4 426.4 4.5 Analytical Gel filtration Day1 EncFtnsH-decamer fractions 20.2 1.8 11.2 EncFtnsH-monomer fractions 2.9 21.9 0.1 Fe(NH4)2(SO4)2/EncFtnsH-monomer 11.0 13.0 0.8 FeSO4-HCl/EncFtnsH-monomer 11.3 11.4 1.0 Analytical Gel filtration Day2 EncFtnsH-monomer fractions 8.3 22.8 0.4 CoCl2/EncFtnsH-monomer 17.7 14.5 1.2 MnCl2/EncFtnsH-monomer 3.1 30.5 0.1 ZnSO4/EncFtnsH-monomer 20.4 9.0 2.3 FeCl3/EncFtnsH-monomer 3.9 28.6 0.1 Analytical Gel filtration Day3 EncFtnsH-monomer fractions 6.3 23.4 0.3 MgSO4/EncFtnsH-monomer 5.8 30.2 0.2 Ca acetate/EncFtnsH-monomer 5.6 25.2 0.2 </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20464</offset><text>We purified EncFtnsH from E. coli grown in MM with or without the addition of 1 mM Fe(NH4)2(SO4)2. The decamer to monomer ratio in the sample purified from cells grown in iron-supplemented media was 4.5, while that from the iron-free media was 0.11, suggesting that iron induces the oligomerization of EncFtnsH in vivo (Figure 3A, Table 3). To test the metal-dependent oligomerization of EncFtnsH in vitro, we incubated the protein with various metal cations and subjected samples to analytical SEC and non-denaturing PAGE. Of the metals tested, only Fe2+, Zn2+ and Co2+ induced the formation of significant amounts of the decamer (Figure 3B, Figure 3—figure supplement 1/2). While Fe2+ induces the multimerization of EncFtnsH, Fe3+ in the form of FeCl3 does not have this effect on the protein, highlighting the apparent preference this protein has for the ferrous form of iron. To determine if the oligomerization of EncFtnsH was concentration dependent we performed analytical SEC at 90 and 700 µM protein concentration (Figure 3C). At the higher concentration, no increase in the decameric form of EncFtn was observed; however, the shift in the major peak from the position of the monomer species indicated a tendency to dimerize at high concentration.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>21725</offset><text>Crystal structure of EncFtnsH</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>21755</offset><text>Electrostatic surface of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>21790</offset><text>The solvent accessible surface of EncFtnsH is shown, colored by electrostatic potential as calculated using the APBS plugin in PyMOL. Negatively charged regions are colored red and positive regions in blue, neutral regions in grey. (A) View of the surface of the EncFtnsH decamer looking down the central axis. (B) Orthogonal view of (A). (C) Cutaway view of (B) showing the charge distribution within the central cavity.</text></passage><passage><infon key="file">elife-18972-fig4-figsupp1.jpg</infon><infon key="id">fig4s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22212</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.013</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>22259</offset><text>Crystal structure of EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22290</offset><text>(A) Overall architecture of EncFtnsH. Transparent solvent accessible surface view with α-helices shown as tubes and bound metal ions as spheres. Alternating subunits are colored blue and green for clarity. The doughnut-like decamer is 7 nm in diameter and 4.5 nm thick. (B) Monomer of EncFtnsH shown as a secondary structure cartoon. (C/D) Dimer interfaces formed in the decameric ring of EncFtnsH. Subunits are shown as secondary structure cartoons and colored blue and green for clarity. Bound metal ions are shown as orange spheres for Fe3+ and grey and white spheres for Ca2+.</text></passage><passage><infon key="file">elife-18972-fig4.jpg</infon><infon key="id">fig4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22875</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.012</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22922</offset><text>We determined the crystal structure of EncFtnsH by molecular replacement to 2.0 Å resolution (see Table 1 for X-ray data collection and refinement statistics). The crystallographic asymmetric unit contained thirty monomers of EncFtn with visible electron density for residues 7 – 96 in each chain. The protein chains were arranged as three identical annular decamers, each with D5 symmetry. The decamer has a diameter of 7 nm and thickness of 4 nm (Figure 4A). The monomer of EncFtn has an N-terminal 310-helix that precedes two 4 nm long antiparallel α-helices arranged with their long axes at 25° to each other; these helices are followed by a shorter 1.4 nm helix projecting at 70° from α2 (Figure 4B). The C-terminal region of the crystallized construct extends from the outer circumference of the ring, indicating that the encapsulin localization sequence in the full-length protein is on the exterior of the ring and is thus free to interact with its binding site on the encapsulin shell protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23933</offset><text>The monomer of EncFtnsH forms two distinct dimer interfaces within the decamer (Figure 4 C/D). The first dimer is formed from two monomers arranged antiparallel to each other, with α1 from each monomer interacting along their lengths and α3 interdigitating with α2 and α3 of the partner chain. This interface buries one third of the surface area from each partner and is stabilized by thirty hydrogen bonds and fourteen salt bridges (Figure 4C). The second dimer interface forms an antiparallel four-helix bundle between helices 1 and 2 from each monomer (Figure 4D). This interface is less extensive than the first and is stabilized by twenty-one hydrogen bonds, six salt bridges, and a number of metal ions.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24659</offset><text>The arrangement of ten monomers in alternating orientation forms the decamer of EncFtn, which assembles as a pentamer of dimers (Figure 4A). Each monomer lies at 45° relative to the vertical central-axis of the ring, with the N-termini of alternating subunits capping the center of the ring at each end, while the C-termini are arranged around the circumference. The central hole in the ring is 2.5 nm at its widest in the center of the complex, and 1.5 nm at its narrowest point near the outer surface, although it should be noted that a number of residues at the N-terminus are not visible in the crystallographic electron density and these may occupy the central channel. The surface of the decamer has distinct negatively charged patches, both within the central hole and on the outer circumference, which form spokes through the radius of the complex (Figure 4—figure supplement 1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>25550</offset><text>EncFtn ferroxidase center</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>25576</offset><text>Putative ligand-binding site in EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>25618</offset><text>(A) Wall-eyed stereo view of the dimer interface of EncFtn. Protein chains are shown as sticks, with 2mFo-DFc electron density shown in blue mesh and contoured at 1.5 σ and mFo-DFc shown in green mesh and contoured at 3 σ. (B) Wall-eyed stereo view of putative metal binding site at the external surface of EncFtnsH. Protein chains and electron density maps are shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig5-figsupp1.jpg</infon><infon key="id">fig5s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26005</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.015</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>26052</offset><text>EncFtnsH metal binding sites.</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26082</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH. Protein residues are shown as sticks with blue and green carbons for the different subunits, iron ions are shown as orange spheres and calcium as grey spheres, and the glycolic acid ligand is shown with yellow carbon atoms coordinated above the di-iron center. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ and the NCS-averaged anomalous difference map is shown as an orange mesh and contoured at 10 σ. (B) Iron coordination within the FOC including residues Glu32, Glu62, His65 and Tyr39 from two chains. Protein and metal ions are shown as in A. Coordination between the protein and iron ions is shown as yellow dashed lines with distances indicated. (C) Coordination of calcium within the dimer interface by four glutamic acid residues (E31 and E34 from two chains). The calcium ion is shown as a grey sphere and water molecules involved in the coordination of the calcium ion are shown as crosses. (D) Metal coordination site on the outer surface of EncFtnsH. The two calcium ions are coordinated by residues His57, Glu61 and Glu64 from the two chains of the FOC dimer, and are located at the outer surface of the complex, positioned 10 Å away from the FOC iron.</text></passage><passage><infon key="file">elife-18972-fig5.jpg</infon><infon key="id">fig5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27371</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.014</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27418</offset><text>The electron density maps of the initial EncFtnsH model displayed significant positive peaks in the mFo-DFc map at the center of the 4-helix bundle dimer (Figure 5—figure supplement 1). Informed by the ICP-MS data indicating the presence of iron in the protein we collected diffraction data at the experimentally determined iron absorption edge (1.74 Å) and calculated an anomalous difference Fourier map using this data. Inspection of this map showed two 10-sigma peaks between residues Glu32, Glu62 and His65 of two adjacent chains, and a statistically smaller 5-sigma peak between residues Glu31 and Glu34 of the two chains. Modeling metal ions into these peaks and refinement of the anomalous scattering parameters allowed us to identify these as two iron ions and a calcium ion respectively (Figure 5A). An additional region of asymmetric electron density near the di-iron binding site in the mFo-DFc map was modeled as glycolic acid, presumably a breakdown product of the PEG 3350 used for crystallization. This di-iron center has an Fe-Fe distance of 3.5 Å, Fe-Glu-O distances between 2.3 and 2.5 Å, and Fe-His-N distances of 2.5 Å (Figure 5B). This coordination geometry is consistent with the di-nuclear ferroxidase center (FOC) found in ferritin. It is interesting to note that although we did not add any additional iron to the crystallization trials, the FOC was fully occupied with iron in the final structure, implying that this site has a very high affinity for iron.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>28907</offset><text>The calcium ion coordinated by Glu31 and Glu34 adopts heptacoordinate geometry, with coordination distances of 2.5 Å between the metal ion and carboxylate oxygens of Glu31 and Glu34 (E31/34-site). A number of ordered solvent molecules are also coordinated to this metal ion at a distance of 2.5 Å. This heptacoordinate geometry is common in crystal structures with calcium ions (Figure 5C). While ICP-MS indicated that there were negligible amounts of calcium in the purified protein, the presence of 140 mM calcium acetate in the crystallization mother liquor favors the coordination of calcium at this site. The fact that the protein does not multimerize in solution in the presence of Fe3+ may indicate that these metal binding sites have a lower affinity for the ferric form of iron, which is the product of the ferroxidase reaction. A number of additional metal-ions were present at the outer circumference of at least one decamer in the asymmetric unit (Figure 5D). These ions are coordinated by His57, Glu61 and Glu64 from both chains in the FOC dimer and are 4.5 Å apart; Fe-Glu-O distances are between 2.5 and 3.5 Å and the Fe-His-N distances are 4 and 4.5 Å.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>30081</offset><text>Comparison of quaternary structure of EncFtnsH and ferritin.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30142</offset><text>(A) Aligned FOC of EncFtnsH and Pseudo-nitzschia multiseries ferritin (PmFtn). The metal binding site residues from two EncFtnsH chains are shown in green and blue, while the PmFtn is shown in orange. Fe2+ in the FOC is shown as orange spheres and Ca2+ in EncFtnsH is shown as a grey sphere. The two-fold symmetry axis of the EncFtn FOC is shown with a grey arrow (B) Cross-section surface view of quaternary structure of EncFtnsH and PmFtn as aligned in (A) (dashed black box). The central channel of EncFtnsH is spatially equivalent to the outer surface of ferritin and its outer surface corresponds to the mineralization surface within ferritin.</text></passage><passage><infon key="file">elife-18972-fig6-figsupp1.jpg</infon><infon key="id">fig6s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30791</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.017</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>30838</offset><text>Comparison of the symmetric metal ion binding site of EncFtnsH and the ferritin FOC.</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>30923</offset><text>(A) Structural alignment of the FOC residues in a dimer of EncFtnsH (green/blue) with a monomer of Pseudo-nitzschia multiseries ferritin (PmFtn) (PDBID: 4ITW) (orange). Iron ions are shown as orange spheres and a single calcium ion as a grey sphere. Residues within the FOC are conserved between EncFtn and ferritin PmFtn, with the exception of residues in the position equivalent to H65’ in the second subunit in the dimer (blue). The site in EncFtn with bound calcium is not present in other family members. (B) Secondary structure of aligned dimeric EncFtnsH and monomeric ferritin highlighting the conserved four-helix bundle. EncFtnsH monomers are shown in green and blue and aligned PmFtn monomer in orange as in A. (C) Cartoon of secondary structure elements in EncFtn dimer and ferritin. In the dimer of EncFtn that forms the FOC, the C-terminus of the first monomer (green) and N-terminus of the second monomer (blue) correspond to the position of the long linker between α2 and α3 in ferritin PmFtn.</text></passage><passage><infon key="file">elife-18972-fig6.jpg</infon><infon key="id">fig6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31941</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.016</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>31988</offset><text>Structural alignment of the di-iron binding site of EncFtnsH to the FOC of Pseudo-nitzschia multiseries ferritin (PmFtn, PDB ID: 4ITW) reveals a striking similarity between the metal binding sites of EncFtnsH and the classical ferritins (Figure 6A). The di-iron site of EncFtnsH is by necessity symmetrical, as it is formed through a dimer interface, while the FOC of ferritin does not have these constraints and varies in different species at a position equivalent to His65 of the second EncFtn monomer in the FOC interface (His65’) (Figure 6A). Structural superimposition of the FOCs of ferritin and EncFtn brings the four-helix bundle of the ferritin fold into close alignment with the EncFtn dimer, showing that the two families of proteins have essentially the same architecture around the di-iron center (Figure 6B). The linker connecting helices 2 and 3 of ferritin is congruent with the start of the C-terminal helix of one EncFtn monomer and the N-terminal 310 helix of the second monomer (Figure 6C).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>33004</offset><text>Mass spectrometry of the EncFtn assembly</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>33045</offset><text>Native IM-MS analysis of the apo-EncFtnsH monomer.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>33096</offset><text>(A) Mass spectrum of apo-EncFtnsH acquired from 100 mM ammonium acetate pH 8.0 under native MS conditions. The charge state distribution observed is bimodal, with peaks corresponding to the 6+ to 15+ charge states of apo-monomer EncFtnsH (neutral average mass 13,194.3 Da). (B) The arrival time distributions (ion mobility data) of all ions in the apo-EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). (B) Right, the arrival time distribution of the 6+ (orange) and 7+ (green) charge state (dashed colored‐box) has been extracted and plotted; The arrival time distributions for these ion is shown (ms), along with the calibrated collision cross section, Ω (nm2). (C) The collision cross section of a single monomer unit from the crystal structure of the Fe-loaded EncFtnsH decamer was calculated to be 15.8 nm2 using IMPACT v. 0.9.1. The +8 to +15 protein charge states have observed CCS between 20–26 nm2, which is significantly higher than the calculated CCS for an EncFtnsH monomer taken from the decameric assembly crystal structure (15.8 nm2). The mobility of the +7 charge state displays broad drift-time distribution with maxima consistent with CCS of 15.9 and 17.9 nm2. Finally, the 6+ charge state of EncFtnsH has mobility consistent with a CCS of 12.3 nm2, indicating a more compact/collapsed structure. It is clear from this data that apo-EncFtnsH exists in several gas phase conformations. The range of charge states occupied by the protein (6+ to 15+) and the range of CCS in which the protein is observed (12.3 nm2 – 26 nm2) are both large. In addition, many of the charge states observed have higher charge than the theoretical maximal charge on spherical globular protein, as determined by the De La Mora relationship (ZR = 0.0778m; for the EncFtnsH monomer ZR = 8.9) Fernandez. As described by Beveridge et al., all these factors are indicative of a disordered protein.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp1.jpg</infon><infon key="id">fig7s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35036</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.019</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>35083</offset><text>Gas-phase disassembly of the holo-EncFtnsH decameric assembly.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>35146</offset><text>The entire charge state distribution of the Fe-loaded holo- EncFtnsH assembly (green circles) was subject to collisional-induced dissociation (CID) by increasing the source cone voltage to 200 V and the trap voltage to 50 V. The resulting CID mass spectrum (A) revealed that dissociation of the holo- EncFtnsH decamer primarily occurred via ejection of a highly charged monomer (blue circles), leaving the ‘stripped’ complex (a 9mer; 118.7 kDa; yellow circles). The mass of the ejected-monomer is consistent with apo- EncFtnsH (13.2 kDa), suggesting unfolding of the monomer (and loss of Fe) occurs during ejection from the complex. This observation of asymmetric charge partitioning of the sub-complexes with respect to the mass of the complex is consistent with the 'typical' pathway of dissociation of protein assemblies by CID, as described by. In addition, a third, lower abundance, charge state distribution is observed which overlaps the EncFtn ejected monomer charge state distribution; this region of the spectrum is highlighted in (B). This distribution is consistent with an ejected EncFtnsH dimer (orange circles). Interestingly, closer analysis of the individual charge state of this dimeric CID product shows that this sub-complex exists in three forms – displaying mass consistent with an EncFtnsH dimer binding 0, 1, and 2 Fe ions. This is highlighted in (C), where the 15+ charge state of the EncFtnsH dimer is shown; 3 peaks are observed with m/z 1760.5, 1763.8, and 1767.0 Th – the lowest peak corresponds to neutral masses of 26392.5 Da [predicted EncFtnsH dimer, (C572H884N172O185S2)2; 26388.6 Da]. The two further peaks have a delta-mass of ~+50 Da, consistent with Fe binding. We interpret these observations as partial ‘atypical’ CID fragmentation of the decameric complex – i.e. fragmentation of the initial complex with retention of subunit and ligand interactions. A schematic summary of these results is displayed in (D). We postulate the high stability of this iron-bound dimer sub-complex is due to the metal coordination at the dimer interface, increasing the strength of the dimer interface. Taken together, these observations support our findings that the topology of the decameric EncFtnsH assembly is arranged as a pentamer of dimers, with two Fe ions at each dimer interface.</text></passage><passage><infon key="file">elife-18972-fig7-figsupp2.jpg</infon><infon key="id">fig7s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37473</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.020</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>37520</offset><text>Native mass spectrometry and ion mobility analysis of iron loading in EncFtnsH.</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>37600</offset><text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Native nanoelectrospray ionization (nESI) mass spectrometry of EncFtnsH at varying iron concentrations. A1, nESI spectrum of iron-free EncFtnsH displays a charge state distribution consistent with EncFtnsH monomer (blue circles, 13,194 Da). Addition of 100 µM (A2) and 300 µM (A3) Fe2+ results in the appearance of a second higher molecular weight charge state distribution consistent with a decameric assembly of EncFtnsH (green circles, 132.6 kDa). (B) Ion mobility (IM)-MS of the iron-bound holo-EncFtnsH decamer. Top, Peaks corresponding to the 22+ to 26+ charge states of a homo-decameric assembly of EncFtnsH are observed (132.6 kDa). Top Insert, Analysis of the 24+ charge state of the assembly at m/z 5528.2 Th. The theoretical average m/z of the 24+ charge state with no additional metals bound is marked by a red line (5498.7 Th); the observed m/z of the 24+ charge state indicates that the EncFtnsH assembly binds between 10 (green line, 5521.1 Th) and 15 Fe ions (blue line, 5532.4 Th) per decamer. Bottom, The arrival time distributions (ion mobility data) of all ions in the EncFtnsH charge state distribution displayed as a greyscale heat map (linear intensity scale). Bottom right, The arrival time distribution of the 24+ charge state (dashed blue box) has been extracted and plotted. The drift time for this ion is shown (ms), along with the calibrated collision cross section (CCS), Ω (nm2).</text></passage><passage><infon key="file">elife-18972-fig7.jpg</infon><infon key="id">fig7</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>39130</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.018</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>39177</offset><text>In order to confirm the assignment of the oligomeric state of EncFtnsH and investigate further the Fe2+-dependent assembly, we used native nano-electrospray ionization (nESI) and ion-mobility mass spectrometry (IM-MS). As described above, by recombinant production of EncFtnsH in minimal media we were able to limit the bioavailability of iron. Native MS analysis of EncFtnsH produced in this way displayed a charge state distribution consistent with an EncFtnsH monomer (blue circles, Figure 7A1) with an average neutral mass of 13,194 Da, in agreement with the predicted mass of the EncFtnsH protein (13,194.53 Da). Under these conditions, no significant higher order assembly was observed and the protein did not have any coordinated metal ions. Titration with Fe2+ directly before native MS analysis resulted in the appearance of a new charge state distribution, consistent with an EncFtnsH decameric assembly (+22 to +26; 132.65 kDa) (Figure 7A2/3). After instrument optimization, the mass resolving power achieved was sufficient to assign iron-loading in the complex to between 10 and 15 Fe ions per decamer (Figure 7B, inset top right), consistent with the presence of 10 irons in the FOC and the coordination of iron in the Glu31/34-site occupied by calcium in the crystal structure (Δmass observed ~0.67 kDa). MS analysis of EncFtnsH after addition of further Fe2+ did not result in iron loading above this stoichiometry. Therefore, the extent of iron binding seen is limited to the FOC and Glu31/34 secondary metal binding site. These data suggest that the decameric assembly of EncFtnsH does not accrue iron in the same manner as classical ferritin, which is able to sequester around 4500 iron ions within its nanocage. Ion mobility analysis of the EncFtnsH decameric assembly, collected with minimal collisional activation, suggested that it consists of a single conformation with a collision cross section (CCS) of 58.2 nm2 (Figure 7B). This observation is in agreement with the calculated CCS of 58.7 nm2derived from our crystal structure of the EncFtnsH decamer. By contrast, IM-MS measurements of the monomeric EncFtnsH at pH 8.0 under the same instrumental conditions revealed that the metal-free protein monomer exists in a wide range of charge states (+6 to +16) and adopts many conformations in the gas phase with collision cross sections ranging from 12 nm2 to 26 nm2 (Figure 7—figure supplement 1). These observations are indicative of an unstructured protein with little secondary or tertiary structure. Thus, IM-MS studies highlight that higher order structure in EncFtnsH is mediated/stabilized by metal binding, an observation that is in agreement with our solution studies. Taken together, these results suggest that di-iron binding, forming the FOC in EncFtnsH, is required to stabilize the 4-helix bundle dimer interface, essentially reconstructing the classical ferritin-like fold; once stabilized, these dimers readily associate as pentamers, and the overall assembly adopts the decameric ring arrangement observed in the crystal structure.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>42254</offset><text>We subsequently performed gas phase disassembly of the decameric EncFtnsH using collision-induced dissociation (CID) tandem mass spectrometry. Under the correct CID conditions, protein assemblies can dissociate with retention of subunit and ligand interactions, and thus provide structurally-informative evidence as to the topology of the original assembly; this has been termed ‘atypical’ dissociation. For EncFtnsH, this atypical dissociation pathway was clearly evident; CID of the EncFtnsH decamer resulted in the appearance of a dimeric EncFtnsH subcomplex containing 0, 1, or 2 iron ions (Figure 7—figure supplement 2). In light of the crystal structure, this observation can be rationalized as dissociation of the EncFtnsH decamer by disruption of the non-FOC interface with at least partial retention of the FOC interface and the FOC-Fe. Thus, this observation supports our crystallographic assignment of the overall topology of the EncFtnsH assembly as a pentameric assembly of dimers with two iron ions located at the FOC dimer interface. In addition, this analysis provides evidence that the overall architecture of the complex is consistent in the crystal, solution and gas phases.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>43454</offset><text>Ferroxidase activity</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>43475</offset><text>TEM visualization of iron-loaded bacterial nanocompartments and ferritin.</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>43549</offset><text>Decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin, at 8.5 µM, were mixed with 147 µM, 1 mM, 1 mM and 215 µM acidic Fe(NH4)2(SO4)2, respectively. Protein mixtures were incubated at room temperature for 1 hr prior to TEM analysis with or without uranyl acetate stain. (A–D) Unstained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 35,000 x magnification and scale bars indicate 100 nm. (E) Protein-free sample as a control. (F–I) Stained EncFtnsH, encapsulin, EncFtn-Enc, apoferritin loaded with Fe2+, respectively, with 140,000 x magnification and scale bars indicate 25 nm.</text></passage><passage><infon key="file">elife-18972-fig8-figsupp1.jpg</infon><infon key="id">fig8s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44180</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.022</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>44227</offset><text>Spectroscopic evidence for the ferroxidase activity and comparison of iron loading capacity of apoferritin, EncFtnsH, encapsulin, and EncFtn-Enc.</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>44373</offset><text>(A) Apoferritin (10 μM monomer concentration) and EncFtnsH decamer fractions (20 μM monomer concentration, 10 μM FOC concentration) were incubated with 20 and 100 μM iron (2 and 10 times molar equivalent Fe2+ per FOC) and progress curves of the oxidation of Fe2+ to Fe3+ at 315 nm were recorded in a spectrophotometer. The background oxidation of iron at 20 and 100 μM in enzyme-free controls are shown for reference. (B) Encapsulin and EncFtn-Enc complexes at 10 μM asymmetric unit concentration were incubated with Fe2+ at 20 and 100 μM and progress curves for iron oxidation at A315 were measured in a UV/visible spectrophotometer. Enzyme free controls for background oxidation of Fe2+ are shown for reference. (C) Histogram of the iron loading capacity per biological assembly of EncFtnsH, encapsulin, EncFtn-Enc and apoferritin. The results shown are for three technical replicates and represent the optimal iron loading by the complexes after three hours when incubated with Fe2+.</text></passage><passage><infon key="file">elife-18972-fig8.jpg</infon><infon key="id">fig8</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>45367</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.021</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>45414</offset><text>In light of the identification of an iron-loaded FOC in the crystal structure of EncFtn and our native mass spectrometry data, we performed ferroxidase and peroxidase assays to demonstrate the catalytic activity of this protein. In addition, we also assayed equine apoferritin, an example of a classical ferritin enzyme, as a positive control. Unlike the Dps family of ferritin-like proteins, EncFtn showed no peroxidase activity when assayed with the substrate ortho-phenylenediamine. The ferroxidase activity of EncFtnsH was measured by recording the progress curve of Fe2+ oxidation to Fe3+ at 315 nm after addition of 20 and 100 µM Fe2+ (2 and 10 times molar ratio Fe2+/FOC). In both experiments the rate of oxidation was faster than background oxidation of Fe2+ by molecular oxygen, and was highest for 100 µM Fe2+ (Figure 8A). These data show that recombinant EncFtnsH acts as an active ferroxidase enzyme. When compared to apoferritin, EncFtnsH oxidized Fe2+ at a slower rate and the reaction did not run to completion over the 1800 s of the experiment. Addition of higher quantities of iron resulted in the formation of a yellow/red precipitate at the end of the reaction. We also performed these assays on purified recombinant encapsulin; which, when assayed alone, did not display ferroxidase activity above background Fe2+ oxidation (Figure 8B). In contrast, complexes of the full EncFtn encapsulin nanocompartment (i.e. the EncFtn-Enc protein complex) displayed ferroxidase activity comparable to apoferritin without the formation of precipitates (Figure 8B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>46994</offset><text>We attributed the precipitates observed in the EncFtnsH ferroxidase assay to the production of insoluble Fe3+ complexes, which led us to propose that EncFtn does not directly store Fe3+ in a mineral form. This observation agrees with native MS results, which indicates a maximum iron loading of 10–15 iron ions per decameric EncFtn; and the structure, which does not possess the enclosed iron-storage cavity characteristic of classical ferritins and Dps family proteins that can directly accrue mineralized Fe3+ within their nanocompartment structures.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>47549</offset><text>To analyze the products of these reactions and determine whether the EncFtn and encapsulin were able to store iron in a mineral form, we performed TEM on the reaction mixtures from the ferroxidase assay. The EncFtnsH reaction mixture showed the formation of large, irregular electron-dense precipitates (Figure 8—figure supplement 1A). A similar distribution of particles was observed after addition of Fe2+ to the encapsulin protein (Figure 8—figure supplement 1B). In contrast, addition of Fe2+ to the EncFtn-Enc nanocompartment resulted in small, highly regular, electron dense particles of approximately 5 nm in diameter (Figure 8—figure supplement 1C); we interpret these observations as controlled mineralization of iron within the nanocompartment. Addition of Fe2+ to apoferritin resulted in a mixture of large particles and small (~2 nm) particles consistent with partial mineralization by the ferritin and some background oxidation of the iron (Figure 8—figure supplement 1D). Negative stain TEM of these samples revealed that upon addition of iron, the EncFtnsH protein showed significant aggregation (Figure 8—figure supplement 1F); while the encapsulin, EncFtn-Enc system, and apoferritin are present as distinct nanocompartments without significant protein aggregation (Figure 8—figure supplement 1G–I).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>48882</offset><text>Iron storage in encapsulin nanocompartments</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>48926</offset><text>The results of the ferroxidase assay and micrographs of the reaction products suggest that the oxidation and mineralization function of the classical ferritins are split between the EncFtn and encapsulin proteins, with the EncFtn acting as a ferroxidase and the encapsulin shell providing an environment and template for iron mineralization and storage. To investigate this further, we added Fe2+ at various concentrations to samples of apo-ferritin, EncFtn, isolated encapsulin, and the EncFtn-Enc protein complex, and subjected these samples to a ferrozine assay to quantify the amount of iron associated with the proteins after three hours of incubation. The maximum iron loading capacity of these systems was calculated as the quantity of iron per biological assembly (Figure 8C). In this assay, the EncFtnsH decamer binds a maximum of around 48 iron ions before excess iron induces protein precipitation. The encapsulin shell protein can sequester about 2200 iron ions before significant protein loss occurs, and the reconstituted EncFtn-Enc nanocompartment sequestered about 4150 iron ions. This latter result is significantly more than the apoferritin used in our assay, which sequesters approximately 570 iron ions in this assay (Figure 8C, Table 5).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>50186</offset><text>Consideration of the functional oligomeric states of these proteins, where EncFtn is a decamer and encapsulin forms an icosahedral cage, and estimation of the iron loading capacity of these complexes gives insight into the role of the two proteins in iron storage and mineralization. EncFtn decamers bind up to 48 iron ions (Figure 8C), which is significantly higher than the stoichiometry of fifteen metal ions visible in the FOC and E31/34-site of the crystal structure of the EncFtnsH decamer and our MS analysis. The discrepancy between these solution measurements and our MS analysis may indicate that there are additional metal-binding sites on the interior channel and exterior faces of the protein; this is consistent with our identification of a number of weak metal-binding sites at the surface of the protein in the crystal structure (Figure 5D). These observations are consistent with hydrated Fe2+ ions being channeled to the active site from the E31/34-site and the subsequent exit of Fe3+ products on the outer surface, as is seen in other ferritin family proteins. While the isolated encapsulin shell does not display any ferroxidase activity, it binds around 2200 iron ions in our assay (Table 5). This implies that the shell can bind a significant amount of iron on its outer and inner surfaces. While the maximum reported loading capacity of classical ferritins is approximately 4500 iron ions, in our assay system we were only able to load apoferritin with around 570 iron ions. However, the recombinant EncFtn-Enc nanocompartment was able to bind over 4100 iron ions in the same time period, over seven times the amount seen for the apoferritin. We note we do not reach the experimental maximum iron loading for apoferritin and therefore the total iron-loading capacity of our system may be significantly higher than in this experimental system.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>52055</offset><text>Taken together, our data show that EncFtn can catalytically oxidize Fe2+ to Fe3+; however, iron binding in EncFtn is limited to the FOC and several surface metal binding sites. In contrast, the encapsulin protein displays no catalytic activity, but has the ability to bind a considerable amount of iron. Finally, the EncFtn-Enc nanocompartment complex retains the catalytic activity of EncFtn, and sequesters iron within the encapsulin shell at a higher level than the isolated components of the system, and at a significantly higher level than the classical ferritins. Furthermore, our recombinant nanocompartments may not have the physiological subunit stoichiometry, and the iron-loading capacity of native nanocompartments is potentially much higher than the level we have observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>52843</offset><text>Mutagenesis of the EncFtnsHferroxidase center</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>52889</offset><text>Purification of recombinant R. rubrum EncFtnsH FOC mutants.</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>52949</offset><text>Single mutants E32A, E62A, and H65A of EncFtnsH produced from E. coli BL21(DE3) cells grown in MM and MM supplemented with iron were subjected to Superdex 200 size-exclusion chromatography. (A) Gel-filtration chromatogram of the E32A mutant form of EncFtnsH resulted in an elution profile with a majority of the protein eluting as the decameric form of the protein and a small proportion of monomer. (B) Gel-filtration chromatograhy of the E62A mutant form of EncFtnsH resulted in an elution profile with a single major decameric peak. (C) Gel-filtration chromatography of the H65A mutant form of EncFtnsH resulted in a single peak corresponding to the protein monomer.</text></passage><passage><infon key="file">elife-18972-fig9.jpg</infon><infon key="id">fig9</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>53626</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.023</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>53673</offset><text>To investigate the structural and biochemical role played by the metal binding residues in the di-iron FOC of EncFtnsH we produced alanine mutations in each of these residues: Glu32, Glu62, and His65. These EncFtnsH mutants were produced in E. coli cells grown in MM, both in the absence and presence of additional iron. The E32A and E62A mutants eluted from SEC at a volume consistent with the decameric form of EncFtnsH, with a small proportion of monomer; the H65A mutant eluted at a volume consistent with the monomeric form of EncFtnsH (Figure 9). For all of the mutants studied, no change in oligomerization state was apparent upon addition of Fe2+ in vitro.</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>54339</offset><text>Native mass spectrometry of EncFtnsH mutants.</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54385</offset><text>All spectra were acquired in 100 mM ammonium acetate, pH 8.0 with a protein concentration of 5 µM. (A) Wild-type EncFtnsH in the absence of iron displays a charge state distribution consistent with a monomer (see also Figure 8). (B) E32A EncFtnsH displays a charge states consistent with a decamer (green circles); a minor species, consistent with the monomer of E32A mutant is also observed (blue circles). (C) E62A EncFtnsH displays charge states consistent with a decamer (green circles). (D) H65A EncFtnsH displays charge states consistent with both monomer (blue circles) and dimer (purple circles).</text></passage><passage><infon key="file">elife-18972-fig10.jpg</infon><infon key="id">fig10</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>54994</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.024</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>55041</offset><text>In addition to SEC studies, native mass spectrometry of the apo-EncFtnsH mutants was performed and compared with the wild-type apo-EncFtnsH protein (Figure 10). As described above, the apo-EncFtnsH has a charge state distribution consistent with an unstructured monomer, and decamer formation is only initiated upon addition of ferrous iron. Both the E32A mutant and E62A mutant displayed charge state distributions consistent with decamers, even in the absence of Fe2+. This gas-phase observation is consistent with SEC measurements, which indicate both of these variants were also decamers in solution. Thus it seems that these mutations allow the decamer to form in the absence of iron in the FOC. In contrast to the glutamic acid mutants, MS analysis of the H65A mutant is similar to wild-type apo-EncFtnsH and is present as a monomer; interestingly a minor population of dimeric H65A was also observed.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>55950</offset><text>We propose that the observed differences in the oligomerization state of the E32A and E62A mutants compared to wild-type are due to the changes in the electrostatic environment within the FOC. At neutral pH the glutamic acid residues are negatively charged, while the histidine residues are predominantly in their uncharged state. In the wild-type (WT) EncFtnsH this leads to electrostatic repulsion between subunits in the absence of iron. Coordination of Fe2+ in this site stabilizes the dimer and reconstitutes the active FOC. The geometric arrangement of Glu32 and Glu62 in the FOC explains their behavior in solution and the gas phase, where they both favor the formation of decamers due to the loss of a repulsive negative charge. The FOC in the H65A mutant is destabilized through the loss of this metal coordinating residue and potential positive charge carrier, thus favoring the monomer in solution and the gas phase.</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>56879</offset><text>Data collection and refinement statistics. Statistics for the highest-resolution shell are shown in parentheses. Friedel mates were averaged when calculating reflection numbers and statistics.</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>57072</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.025</text></passage><passage><infon key="file">tbl4.xml</infon><infon key="id">tbl4</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"/><th rowspan="1" colspan="1"><styled-content style="color: #000000;">WT</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">E32A</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">E62A</styled-content></th><th rowspan="1" colspan="1"><styled-content style="color: #000000;">H65A</styled-content></th></tr></thead><tbody><tr><td rowspan="1" colspan="1">Data collection</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">Wavelength (Å)</td><td rowspan="1" colspan="1">1.74</td><td rowspan="1" colspan="1">1.73</td><td rowspan="1" colspan="1">1.73</td><td rowspan="1" colspan="1">1.74</td></tr><tr><td rowspan="1" colspan="1">Resolution range (Å)</td><td rowspan="1" colspan="1">49.63 - 2.06 <break/>(2.10 - 2.06)</td><td rowspan="1" colspan="1">48.84 - 2.59 <break/>(2.683 - 2.59)</td><td rowspan="1" colspan="1">48.87 - 2.21 <break/>(2.29 - 2.21)</td><td rowspan="1" colspan="1">48.86 - 2.97 <break/>(3.08 - 2.97)</td></tr><tr><td rowspan="1" colspan="1">Space group</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td><td rowspan="1" colspan="1"><italic>P</italic> 1 2<sub>1</sub> 1</td></tr><tr><td rowspan="1" colspan="1">Unit cell (Å) <italic>a</italic>
<break/> b <break/>
<italic> c</italic>
<break/> β (°)</td><td rowspan="1" colspan="1">98.18 <break/>120.53 <break/>140.30 <break/>95.36</td><td rowspan="1" colspan="1">97.78 <break/>120.28 <break/>140.53 <break/>95.41</td><td rowspan="1" colspan="1">98.09 <break/>120.23 <break/>140.36 <break/>95.50</td><td rowspan="1" colspan="1">98.03 <break/>120.29 <break/>140.43 <break/>95.39</td></tr><tr><td rowspan="1" colspan="1">Total reflections</td><td rowspan="1" colspan="1">1,264,922 <break/>(41,360)</td><td rowspan="1" colspan="1">405,488 <break/>(36,186)</td><td rowspan="1" colspan="1">1,069,345 <break/>(95,716)</td><td rowspan="1" colspan="1">323,853 <break/>(32,120)</td></tr><tr><td rowspan="1" colspan="1">Unique reflections</td><td rowspan="1" colspan="1">197,873 <break/>(8,766)</td><td rowspan="1" colspan="1">100,067 <break/>(9,735)</td><td rowspan="1" colspan="1">162,379 <break/>(15,817)</td><td rowspan="1" colspan="1">66,658 <break/>(6,553)</td></tr><tr><td rowspan="1" colspan="1">Multiplicity</td><td rowspan="1" colspan="1">6.4 (4.7)</td><td rowspan="1" colspan="1">4.1 (3.7)</td><td rowspan="1" colspan="1">6.6 (6.1)</td><td rowspan="1" colspan="1">4.9 (4.9)</td></tr><tr><td rowspan="1" colspan="1">Anomalous multiplicity</td><td rowspan="1" colspan="1">3.2 (2.6)</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td></tr><tr><td rowspan="1" colspan="1">Completeness (%)</td><td rowspan="1" colspan="1">99.2 (88.6)</td><td rowspan="1" colspan="1">99.0 (97.0)</td><td rowspan="1" colspan="1">100 (97.0)</td><td rowspan="1" colspan="1">100 (99.0)</td></tr><tr><td rowspan="1" colspan="1">Anomalous completeness (%)</td><td rowspan="1" colspan="1">96.7 (77.2)</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td><td rowspan="1" colspan="1">N/A</td></tr><tr><td rowspan="1" colspan="1">Mean I/sigma(I)</td><td rowspan="1" colspan="1">10.6 (1.60)</td><td rowspan="1" colspan="1">8.46 (1.79)</td><td rowspan="1" colspan="1">13.74 (1.80)</td><td rowspan="1" colspan="1">8.09 (1.74)</td></tr><tr><td rowspan="1" colspan="1">Wilson B-factor</td><td rowspan="1" colspan="1">26.98</td><td rowspan="1" colspan="1">40.10</td><td rowspan="1" colspan="1">33.97</td><td rowspan="1" colspan="1">52.20</td></tr><tr><td rowspan="1" colspan="1">R<sub>merge</sub></td><td rowspan="1" colspan="1">0.123 (0.790)</td><td rowspan="1" colspan="1">0.171 (0.792)</td><td rowspan="1" colspan="1">0.0979 (1.009)</td><td rowspan="1" colspan="1">0.177 (0.863)</td></tr><tr><td rowspan="1" colspan="1">R<sub>meas</sub></td><td rowspan="1" colspan="1">0.147 (0.973)</td><td rowspan="1" colspan="1">0.196 (0.923)</td><td rowspan="1" colspan="1">0.1064 (1.107)</td><td rowspan="1" colspan="1">0.199 (0.966)</td></tr><tr><td rowspan="1" colspan="1">CC1/2</td><td rowspan="1" colspan="1">0.995 (0.469)</td><td rowspan="1" colspan="1">0.985 (0.557)</td><td rowspan="1" colspan="1">0.998 (0.642)</td><td rowspan="1" colspan="1">0.989 (0.627)</td></tr><tr><td rowspan="1" colspan="1">CC*</td><td rowspan="1" colspan="1">0.999 (0.846)</td><td rowspan="1" colspan="1">0.996 (0.846)</td><td rowspan="1" colspan="1">0.999 (0.884)</td><td rowspan="1" colspan="1">0.997 (0.878)</td></tr><tr><td rowspan="1" colspan="1">Image DOI</td><td rowspan="1" colspan="1">10.7488/ds/1342</td><td rowspan="1" colspan="1">10.7488/ds/1419</td><td rowspan="1" colspan="1">10.7488/ds/1420</td><td rowspan="1" colspan="1">10.7488/ds/1421</td></tr><tr><td rowspan="1" colspan="1">Refinement</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">R<sub>work</sub></td><td rowspan="1" colspan="1">0.171 (0.318)</td><td rowspan="1" colspan="1">0.183 (0.288)</td><td rowspan="1" colspan="1">0.165 (0.299)</td><td rowspan="1" colspan="1">0.186 (0.273)</td></tr><tr><td rowspan="1" colspan="1">R<sub>free</sub></td><td rowspan="1" colspan="1">0.206 (0.345)</td><td rowspan="1" colspan="1">0.225 (0351)</td><td rowspan="1" colspan="1">0.216 (0.364)</td><td rowspan="1" colspan="1">0.237 (0.325)</td></tr><tr><td rowspan="1" colspan="1">Number of non-hydrogen atoms</td><td rowspan="1" colspan="1">23,222</td><td rowspan="1" colspan="1">22,366</td><td rowspan="1" colspan="1">22,691</td><td rowspan="1" colspan="1">22,145</td></tr><tr><td rowspan="1" colspan="1">macromolecules</td><td rowspan="1" colspan="1">22,276</td><td rowspan="1" colspan="1">22,019</td><td rowspan="1" colspan="1">21,965</td><td rowspan="1" colspan="1">22,066</td></tr><tr><td rowspan="1" colspan="1">ligands</td><td rowspan="1" colspan="1">138</td><td rowspan="1" colspan="1">8</td><td rowspan="1" colspan="1">24</td><td rowspan="1" colspan="1">74</td></tr><tr><td rowspan="1" colspan="1">water</td><td rowspan="1" colspan="1">808</td><td rowspan="1" colspan="1">339</td><td rowspan="1" colspan="1">702</td><td rowspan="1" colspan="1">5</td></tr><tr><td rowspan="1" colspan="1">Protein residues</td><td rowspan="1" colspan="1">2,703</td><td rowspan="1" colspan="1">2,686</td><td rowspan="1" colspan="1">2,675</td><td rowspan="1" colspan="1">2,700</td></tr><tr><td rowspan="1" colspan="1">RMS(bonds) (Å)</td><td rowspan="1" colspan="1">0.012</td><td rowspan="1" colspan="1">0.005</td><td rowspan="1" colspan="1">0.011</td><td rowspan="1" colspan="1">0.002</td></tr><tr><td rowspan="1" colspan="1">RMS(angles) (°)</td><td rowspan="1" colspan="1">1.26</td><td rowspan="1" colspan="1">0.58</td><td rowspan="1" colspan="1">1.02</td><td rowspan="1" colspan="1">0.40</td></tr><tr><td rowspan="1" colspan="1">Ramachandran favored (%)</td><td rowspan="1" colspan="1">100</td><td rowspan="1" colspan="1">99</td><td rowspan="1" colspan="1">100</td><td rowspan="1" colspan="1">99</td></tr><tr><td rowspan="1" colspan="1">Ramachandran allowed (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">1</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">1</td></tr><tr><td rowspan="1" colspan="1">Ramachandran outliers (%)</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">0</td></tr><tr><td rowspan="1" colspan="1">Clash score</td><td rowspan="1" colspan="1">1.42</td><td rowspan="1" colspan="1">1.42</td><td rowspan="1" colspan="1">1.79</td><td rowspan="1" colspan="1">0.97</td></tr><tr><td rowspan="1" colspan="1">Average B-factor (Å<sup>2</sup>)</td><td rowspan="1" colspan="1">33.90</td><td rowspan="1" colspan="1">42.31</td><td rowspan="1" colspan="1">41.34</td><td rowspan="1" colspan="1">47.68</td></tr><tr><td rowspan="1" colspan="1">macromolecules</td><td rowspan="1" colspan="1">33.80</td><td rowspan="1" colspan="1">42.35</td><td rowspan="1" colspan="1">41.31</td><td rowspan="1" colspan="1">47.60</td></tr><tr><td rowspan="1" colspan="1">ligands</td><td rowspan="1" colspan="1">40.40</td><td rowspan="1" colspan="1">72.80</td><td rowspan="1" colspan="1">65.55</td><td rowspan="1" colspan="1">72.34</td></tr><tr><td rowspan="1" colspan="1">solvent</td><td rowspan="1" colspan="1">36.20</td><td rowspan="1" colspan="1">38.95</td><td rowspan="1" colspan="1">41.46</td><td rowspan="1" colspan="1">33.85</td></tr><tr><td rowspan="1" colspan="1">PDB ID</td><td rowspan="1" colspan="1">5DA5</td><td rowspan="1" colspan="1">5L89</td><td rowspan="1" colspan="1">5L8B</td><td rowspan="1" colspan="1">5L8G</td></tr></tbody></table>
</infon><offset>57119</offset><text> WT E32A E62A H65A Data collection Wavelength (Å) 1.74 1.73 1.73 1.74 Resolution range (Å) 49.63 - 2.06 (2.10 - 2.06) 48.84 - 2.59 (2.683 - 2.59) 48.87 - 2.21 (2.29 - 2.21) 48.86 - 2.97 (3.08 - 2.97) Space group P 1 21 1 P 1 21 1 P 1 21 1 P 1 21 1 Unit cell (Å) a b c β (°) 98.18 120.53 140.30 95.36 97.78 120.28 140.53 95.41 98.09 120.23 140.36 95.50 98.03 120.29 140.43 95.39 Total reflections 1,264,922 (41,360) 405,488 (36,186) 1,069,345 (95,716) 323,853 (32,120) Unique reflections 197,873 (8,766) 100,067 (9,735) 162,379 (15,817) 66,658 (6,553) Multiplicity 6.4 (4.7) 4.1 (3.7) 6.6 (6.1) 4.9 (4.9) Anomalous multiplicity 3.2 (2.6) N/A N/A N/A Completeness (%) 99.2 (88.6) 99.0 (97.0) 100 (97.0) 100 (99.0) Anomalous completeness (%) 96.7 (77.2) N/A N/A N/A Mean I/sigma(I) 10.6 (1.60) 8.46 (1.79) 13.74 (1.80) 8.09 (1.74) Wilson B-factor 26.98 40.10 33.97 52.20 Rmerge 0.123 (0.790) 0.171 (0.792) 0.0979 (1.009) 0.177 (0.863) Rmeas 0.147 (0.973) 0.196 (0.923) 0.1064 (1.107) 0.199 (0.966) CC1/2 0.995 (0.469) 0.985 (0.557) 0.998 (0.642) 0.989 (0.627) CC* 0.999 (0.846) 0.996 (0.846) 0.999 (0.884) 0.997 (0.878) Image DOI 10.7488/ds/1342 10.7488/ds/1419 10.7488/ds/1420 10.7488/ds/1421 Refinement Rwork 0.171 (0.318) 0.183 (0.288) 0.165 (0.299) 0.186 (0.273) Rfree 0.206 (0.345) 0.225 (0351) 0.216 (0.364) 0.237 (0.325) Number of non-hydrogen atoms 23,222 22,366 22,691 22,145 macromolecules 22,276 22,019 21,965 22,066 ligands 138 8 24 74 water 808 339 702 5 Protein residues 2,703 2,686 2,675 2,700 RMS(bonds) (Å) 0.012 0.005 0.011 0.002 RMS(angles) (°) 1.26 0.58 1.02 0.40 Ramachandran favored (%) 100 99 100 99 Ramachandran allowed (%) 0 1 0 1 Ramachandran outliers (%) 0 0 0 0 Clash score 1.42 1.42 1.79 0.97 Average B-factor (Å2) 33.90 42.31 41.34 47.68 macromolecules 33.80 42.35 41.31 47.60 ligands 40.40 72.80 65.55 72.34 solvent 36.20 38.95 41.46 33.85 PDB ID 5DA5 5L89 5L8B 5L8G </text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>59111</offset><text>Iron loading capacity of EncFtn, encapsulin and ferritin. Protein samples (at 8.5 µM) including decameric EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were mixed with Fe(NH4)2(SO4) (in 0.1% (v/v) HCl) of different concentrations in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl buffer at room temperature for 3 hrs in the air. Protein-Fe mixtures were centrifuged at 13,000 x g to remove precipitated material and desalted prior to the Fe and protein content analysis by ferrozine assay and BCA microplate assay, respectively. Fe to protein ratio was calculated to indicate the Fe binding capacity of the protein. Protein stability was compromised at high iron concentrations; therefore, the highest iron loading with the least protein precipitation was used to derive the maximum iron loading capacity per biological assembly (underlined and highlighted in bold). The biological unit assemblies are a decamer for EncFtnsH, a 60mer for encapsulin, a 60mer of encapsulin loaded with 12 copies of decameric EncFtn in the complex, and 24mer for horse spleen apoferritin. Errors are quoted as the standard deviation of three technical repeats in both the ferrozine and BCA microplate assays. The proteins used in Fe loading experiment came from a single preparation.</text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>60373</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.026</text></passage><passage><infon key="file">tbl5.xml</infon><infon key="id">tbl5</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1">Protein sample</th><th rowspan="1" colspan="1">Fe(NH<sub>4)2</sub>(SO<sub>4)2</sub> loading (µM)</th><th rowspan="1" colspan="1">Fe detected by ferrozine assay (µM)</th><th rowspan="1" colspan="1">Protein detected by BCA microplate assay (µM)</th><th rowspan="1" colspan="1">Fe / monomeric protein</th><th rowspan="1" colspan="1">Maximum Fe loading per biological assembly unit</th></tr></thead><tbody><tr><td rowspan="6" colspan="1">8.46 µM EncFtn<sub>sH</sub>-10mer</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">4.73 ± 2.32</td><td rowspan="1" colspan="1">5.26 ± 0.64</td><td rowspan="1" colspan="1">0.90 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">39.9</td><td rowspan="1" colspan="1">9.93 ± 1.20</td><td rowspan="1" colspan="1">5.36 ± 0.69</td><td rowspan="1" colspan="1">1.85 ± 0.22</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">84</td><td rowspan="1" colspan="1">17.99 ± 2.01</td><td rowspan="1" colspan="1">4.96 ± 0.04</td><td rowspan="1" colspan="1">3.63 ± 0.41</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">147</td><td rowspan="1" colspan="1">21.09 ± 1.94</td><td rowspan="1" colspan="1">4.44 ± 0.21</td><td rowspan="1" colspan="1">4.75 ± 0.44</td><td rowspan="1" colspan="1">48 ± 4</td></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">28.68 ± 0.30</td><td rowspan="1" colspan="1">3.73 ± 0.53</td><td rowspan="1" colspan="1">7.68 ± 0.08</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">11.27 ± 1.10</td><td rowspan="1" colspan="1">2.50 ± 0.05</td><td rowspan="1" colspan="1">4.51 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="8" colspan="1">8.50 µM Encapsulin</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">-1.02 ± 0.54</td><td rowspan="1" colspan="1">8.63 ± 0.17</td><td rowspan="1" colspan="1">-0.12 ± 0.06</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">62.24 ± 2.49</td><td rowspan="1" colspan="1">10.01 ± 0.58</td><td rowspan="1" colspan="1">6.22 ± 0.35</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">67.94 ± 3.15</td><td rowspan="1" colspan="1">8.69 ± 0.42</td><td rowspan="1" colspan="1">7.81 ± 0.36</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">450</td><td rowspan="1" colspan="1">107.96 ± 8.88</td><td rowspan="1" colspan="1">8.50 ± 0.69</td><td rowspan="1" colspan="1">12.71 ± 1.05</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">700</td><td rowspan="1" colspan="1">97.51 ± 3.19</td><td rowspan="1" colspan="1">7.26 ± 0.20</td><td rowspan="1" colspan="1">13.44 ± 0.44</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1000</td><td rowspan="1" colspan="1">308.63 ± 2.06</td><td rowspan="1" colspan="1">8.42 ± 0.34</td><td rowspan="1" colspan="1">36.66 ± 0.24</td><td rowspan="1" colspan="1">2199 ± 15</td></tr><tr><td rowspan="1" colspan="1">1500</td><td rowspan="1" colspan="1">57.09 ± 0.90</td><td rowspan="1" colspan="1">1.44 ± 0.21</td><td rowspan="1" colspan="1">39.77 ± 0.62</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">2000</td><td rowspan="1" colspan="1">9.2 ± 1.16</td><td rowspan="1" colspan="1">0.21 ± 0.14</td><td rowspan="1" colspan="1">44.73 ± 5.63</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="8" colspan="1">8.70 µM EncFtn-Enc</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">3.31 ± 1.57</td><td rowspan="1" colspan="1">6.85 ± 0.07</td><td rowspan="1" colspan="1">0.48 ± 0.23</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">224</td><td rowspan="1" colspan="1">116.27 ± 3.74</td><td rowspan="1" colspan="1">7.63 ± 0.12</td><td rowspan="1" colspan="1">15.25 ± 0.49</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">301</td><td rowspan="1" colspan="1">132.86 ± 4.03</td><td rowspan="1" colspan="1">6.66 ± 0.31</td><td rowspan="1" colspan="1">19.96 ± 0.61</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">450</td><td rowspan="1" colspan="1">220.57 ± 27.33</td><td rowspan="1" colspan="1">6.12 ± 1.07</td><td rowspan="1" colspan="1">36.06 ± 4.47</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">700</td><td rowspan="1" colspan="1">344.03 ± 40.38</td><td rowspan="1" colspan="1">6.94 ± 0.17</td><td rowspan="1" colspan="1">49.58 ± 5.82</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1000</td><td rowspan="1" colspan="1">496.00 ± 38.48</td><td rowspan="1" colspan="1">7.19 ± 0.08</td><td rowspan="1" colspan="1">68.94 ± 5.35</td><td rowspan="1" colspan="1">4137 ± 321</td></tr><tr><td rowspan="1" colspan="1">1500</td><td rowspan="1" colspan="1">569.98 ± 73.63</td><td rowspan="1" colspan="1">5.73 ± 0.03</td><td rowspan="1" colspan="1">99.44 ± 12.84</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">2000</td><td rowspan="1" colspan="1">584.30 ± 28.33</td><td rowspan="1" colspan="1">4.88 ± 0.22</td><td rowspan="1" colspan="1">119.62 ± 5.80</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="6" colspan="1">8.50 µM Apoferritin</td><td rowspan="1" colspan="1">0</td><td rowspan="1" colspan="1">3.95 ± 2.26</td><td rowspan="1" colspan="1">9.37 ± 0.24</td><td rowspan="1" colspan="1">0.42 ± 0.25</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">42.5</td><td rowspan="1" colspan="1">10.27 ± 1.12</td><td rowspan="1" colspan="1">8.27 ± 0.30</td><td rowspan="1" colspan="1">1.24 ± 0.18</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">212.5</td><td rowspan="1" colspan="1">44.48 ± 2.76</td><td rowspan="1" colspan="1">7.85 ± 0.77</td><td rowspan="1" colspan="1">5.67 ± 0.83</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">637.5</td><td rowspan="1" colspan="1">160.93 ± 4.27</td><td rowspan="1" colspan="1">6.76 ± 0.81</td><td rowspan="1" colspan="1">23.79 ± 3.12</td><td rowspan="1" colspan="1">571 ± 75</td></tr><tr><td rowspan="1" colspan="1">1275</td><td rowspan="1" colspan="1">114.92 ± 3.17</td><td rowspan="1" colspan="1">3.84 ± 0.30</td><td rowspan="1" colspan="1">29.91 ± 2.95</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">1700</td><td rowspan="1" colspan="1">91.40 ± 3.37</td><td rowspan="1" colspan="1">3.14 ± 0.35</td><td rowspan="1" colspan="1">29.13 ± 3.86</td><td rowspan="1" colspan="1"/></tr></tbody></table>
</infon><offset>60420</offset><text>Protein sample Fe(NH4)2(SO4)2 loading (µM) Fe detected by ferrozine assay (µM) Protein detected by BCA microplate assay (µM) Fe / monomeric protein Maximum Fe loading per biological assembly unit 8.46 µM EncFtnsH-10mer 0 4.73 ± 2.32 5.26 ± 0.64 0.90 ± 0.44 39.9 9.93 ± 1.20 5.36 ± 0.69 1.85 ± 0.22 84 17.99 ± 2.01 4.96 ± 0.04 3.63 ± 0.41 147 21.09 ± 1.94 4.44 ± 0.21 4.75 ± 0.44 48 ± 4 224 28.68 ± 0.30 3.73 ± 0.53 7.68 ± 0.08 301 11.27 ± 1.10 2.50 ± 0.05 4.51 ± 0.44 8.50 µM Encapsulin 0 -1.02 ± 0.54 8.63 ± 0.17 -0.12 ± 0.06 224 62.24 ± 2.49 10.01 ± 0.58 6.22 ± 0.35 301 67.94 ± 3.15 8.69 ± 0.42 7.81 ± 0.36 450 107.96 ± 8.88 8.50 ± 0.69 12.71 ± 1.05 700 97.51 ± 3.19 7.26 ± 0.20 13.44 ± 0.44 1000 308.63 ± 2.06 8.42 ± 0.34 36.66 ± 0.24 2199 ± 15 1500 57.09 ± 0.90 1.44 ± 0.21 39.77 ± 0.62 2000 9.2 ± 1.16 0.21 ± 0.14 44.73 ± 5.63 8.70 µM EncFtn-Enc 0 3.31 ± 1.57 6.85 ± 0.07 0.48 ± 0.23 224 116.27 ± 3.74 7.63 ± 0.12 15.25 ± 0.49 301 132.86 ± 4.03 6.66 ± 0.31 19.96 ± 0.61 450 220.57 ± 27.33 6.12 ± 1.07 36.06 ± 4.47 700 344.03 ± 40.38 6.94 ± 0.17 49.58 ± 5.82 1000 496.00 ± 38.48 7.19 ± 0.08 68.94 ± 5.35 4137 ± 321 1500 569.98 ± 73.63 5.73 ± 0.03 99.44 ± 12.84 2000 584.30 ± 28.33 4.88 ± 0.22 119.62 ± 5.80 8.50 µM Apoferritin 0 3.95 ± 2.26 9.37 ± 0.24 0.42 ± 0.25 42.5 10.27 ± 1.12 8.27 ± 0.30 1.24 ± 0.18 212.5 44.48 ± 2.76 7.85 ± 0.77 5.67 ± 0.83 637.5 160.93 ± 4.27 6.76 ± 0.81 23.79 ± 3.12 571 ± 75 1275 114.92 ± 3.17 3.84 ± 0.30 29.91 ± 2.95 1700 91.40 ± 3.37 3.14 ± 0.35 29.13 ± 3.86 </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>62115</offset><text>To understand the impact of the mutants on the organization and metal binding of the FOC, we determined the X-ray crystal structures of each of the EncFtnsH mutants (See Table 4 for data collection and refinement statistics). The crystal packing of all of the mutants in this study is essentially isomorphous to the EncFtnsH structure. All of the mutants display the same decameric arrangement in the crystals as the EncFtnsH structure, and the monomers superimpose with an average RMSDCα of less than 0.2 Å.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>62628</offset><text>FOC dimer interface of EncFtnsH-E32A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>62673</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E32A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. (B) Views of the FOC of the EncFtnsH-E32Amutant. Protein atoms shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp1.jpg</infon><infon key="id">fig11s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63016</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.028</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63063</offset><text>FOC dimer interface of EncFtnsH-E62A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63108</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-E62A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The single coordinated calcium ion is shown as a grey sphere. (B) Views of the FOC of the EncFtnsH-E62A mutant. Protein atoms shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp2.jpg</infon><infon key="id">fig11s2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63514</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.029</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63561</offset><text>FOC dimer interface of EncFtnsH-H65A mutant.</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63606</offset><text>(A) Wall-eyed stereo view of the metal-binding dimerization interface of EncFtnsH-H65A. Protein residues are shown as sticks with blue and green carbons for the different subunits. The 2mFo-DFc electron density map is shown as a blue mesh contoured at 1.5 σ. The coordinated calcium ions are shown as a grey spheres with coordination distances in the FOC highlighted with yellow dashed lines. (B) Views of the FOC of the EncFtnsH-H65A mutant. Protein atoms and metal ions shown as in (A).</text></passage><passage><infon key="file">elife-18972-fig11-figsupp3.jpg</infon><infon key="id">fig11s3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64099</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.030</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>64146</offset><text>Comparison of the EncFtnsH FOC mutants vs wild type.</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64199</offset><text>The structures of the three EncFtnsH mutants were all determined by X-ray crystallography. The E32A, E62A and H65A mutants were crystallized in identical conditions to the wild type. EncFtnsH structure and were essentially isomorphous in terms of their unit cell dimensions. The FOC residues of the mutants and native EncFtnsH structures are shown as sticks with coordinated Fe2+ as orange and Ca2+ as grey spheres and are colored as follows: wild type, grey; E32A, pink; E62A, green; H65A, blue. Of the mutants, only H65A has any coordinated metal ions, which appear to be calcium ions from the crystallization condition. The overall organization of FOC residues is retained in the mutants, with almost no backbone movements. Significant differences center around Tyr39, which moves to coordinate the bound calcium ions in the H65A mutant; and Glu32, which moves away from the metal ions in this structure.</text></passage><passage><infon key="file">elife-18972-fig11.jpg</infon><infon key="id">fig11</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>65108</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.027</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>65155</offset><text>Close inspection of the region of the protein around the FOC in each of the mutants highlights their effect on metal binding (Figure 11 and Figure 11—figure supplement 1–3). In the E32A mutant the position of the side chains of the remaining iron coordinating residues in the FOC is essentially unchanged, but the absence of the axial-metal coordinating ligand provided by the Glu32 side chain abrogates metal binding in this site. The Glu31/34-site also lacks metal, with the side chain of Glu31 rotated by 180° at the Cβ in the absence of metal (Figure 11—figure supplement 1). The E62A mutant has a similar effect on the FOC to the E32A mutant, however the entry site still has a calcium ion coordinated between residues Glu31 and Glu34 (Figure 11—figure supplement 2). The H65A mutant diverges significantly from the wild type in the position of the residues Glu32 and Tyr39 in the FOC. E32 appears in either the original orientation as the wild type and coordinates Ca2+ in this position, or it is flipped by 180° at the Cβ, moving away from the coordinated calcium ion in the FOC. Tyr39 moves closer to Ca2+ compared to the wild-type and coordinates the calcium ion (Figure 11—figure supplement 3). A single calcium ion is present in the entry site of this mutant; however, Glu31 of one chain is rotated away from the metal ion and is not involved in coordination.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>66541</offset><text>Taken together the results of our data show that these changes to the FOC of EncFtn still permit the formation of the decameric form of the protein. While the proteins all appear decameric in crystals, their solution and gas-phase behavior differs considerably and the mutants no longer show metal-dependent oligomerization. These results highlight the importance of metal coordination in the FOC for the stability and assembly of the EncFtn protein.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>66992</offset><text>Progress curves recording ferroxidase activity of EncFtnsH mutants. 20 µM wild-type EncFtnsH, E32A, E62A and H65A mutants were mixed with 20 µM or 100 µM acidic Fe(NH4)2(SO4)2, respectively.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67195</offset><text>Absorbance at 315 nm was recorded for 1800 s at 25°C as an indication of Fe3+ formation. Protein free samples (dashed and dotted lines) were measured for Fe2+ background oxidation as controls. Assays were performed with three technical repeats. Error bars were showed in shadows behind each curves.</text></passage><passage><infon key="file">elife-18972-fig12-figsupp1.jpg</infon><infon key="id">fig12s1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67495</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.032</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>67542</offset><text>Relative ferroxidase activity of EncFtnsH mutants.</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>67593</offset><text>EncFtnsH, and the mutant forms E32A, E62A and H65A, each at 20 µM, were mixed with 100 µM acidic Fe(NH4)2(SO4)2. Ferroxidase activity of the mutant forms is determined by measuring the absorbance at 315 nm for 1800 s at 25 °C as an indication of Fe3+ formation. The relative ferroxidase activity of mutants is plotted as a proportion of the activity of the wild-type protein using the endpoint measurement of A315. Three technical repeats were performed and the plotted error bars represent the calculated standard deviations. The FOC mutants showed reduced ferroxidase activity to varied extents, among which E62A significantly abrogated the ferroxidase activity.</text></passage><passage><infon key="file">elife-18972-fig12.jpg</infon><infon key="id">fig12</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>68266</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.031</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>68313</offset><text>To address the question of how mutagenesis of the iron coordinating residues affects the enzymatic activity of the EncFtnsH protein we recorded progress curves for the oxidation of Fe2+ to Fe3+ by the different mutants as before. Mutagenesis of E32A and H65A reduces the activity of EncFtnsH by about 40%-55%; the E62A mutant completely abrogates activity, presumably through the loss of the bridging coordination for the formation of the di-nuclear iron center of the FOC (Figure 12). Collectively, the effect of mutating these residues in the FOC confirms the importance of the iron coordinating residues for the ferroxidase activity of the EncFtnsH protein.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>68975</offset><text>Discussion</text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>68986</offset><text>Phylogenetic tree of ferritin family proteins.</text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>69033</offset><text>The tree was built using the Neighbor-Joining method based on step-wise amino acid sequence alignment of the four-helical bundle portions of ferritin family proteins (Supplementary file 1). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree; the likely root of the tree is indicated by a red arrow. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 2.5). The analysis involved 104 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 262 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 </text></passage><passage><infon key="file">elife-18972-fig13.jpg</infon><infon key="id">fig13</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>69870</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.033</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>69917</offset><text>Our study reports on a new class of ferritin-like proteins (EncFtn), which are associated with bacterial encapsulin nanocompartments (Enc). By studying the EncFtn from R. rubrum we demonstrate that iron binding results in assembly of EncFtn decamers, which display a unique annular architecture. Despite a radically different quaternary structure to the classical ferritins, the four-helical bundle scaffold and FOC of EncFtnsH are strikingly similar to ferritin (Figure 6A). A sequence-based phylogenetic tree for proteins in the ferritin family was constructed; in addition to the classical ferritins, bacterioferritins and Dps proteins, our analysis included the encapsulin-associated ferritin-like proteins (EncFtns) and a group related to these, but lacking the encapsulin sequence (Non-EncFtn). The analysis revealed that the EncFtn and Non-EncFtn proteins form groups distinct from the other clearly delineated groups of ferritins, and represent outliers in the tree (Figure 13). While it is difficult to infer ancestral lineages in protein families, the similarity seen in the active site scaffold of these proteins highlights a shared evolutionary relationship between EncFtn proteins and other members of the ferritin superfamily that has been noted in previous studies (; ). From this analysis, we propose that the four-helical fold of the classical ferritins may have arisen through gene duplication of an ancestor of EncFtn. This gene duplication would result in the C-terminal region of one EncFtn monomer being linked to the N-terminus of another and thus stabilizing the four-helix bundle fold within a single polypeptide chain (Figure 6B). Linking the protein together in this way relaxes the requirement for the maintenance of a symmetrical FOC and thus provides a path to the diversity in active-site residues seen across the ferritin family (Figure 6A, residues Glu95, Gln128 and Glu131 in PmFtn, Supplementary file 1).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_2</infon><offset>71858</offset><text>Relationship between ferritin structure and activity</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>71911</offset><text>The quaternary arrangement of classical ferritins into an octahedral nanocage and Dps into a dodecamer is absolutely required for their function as iron storage compartments. The oxidation and mineralization of iron must be spatially separated from the host cytosol to prevent the formation of damaging hydroxyl radicals in the Fenton and Haber-Weiss reactions. This is achieved in all ferritins by confining the oxidation of iron to the interior of the protein complex, thus achieving sequestration of the Fe3+ mineralization product. A structural alignment of the FOC of EncFtn with the classical ferritin PmFtn shows that the central ring of EncFtn corresponds to the external surface of ferritin, while the outer circumference of EncFtn is congruent with the inner mineralization surface of ferritin (Figure 6—figure supplement 1A). This overlay highlights the fact that the ferroxidase center of EncFtn faces in the opposite direction relative to the classical ferritins and is essentially inside out regarding iron storage space (Figure 6—figure supplement 1B, boxed region). Analysis of each of the single mutations (E32A, E62A and H65A) made in the FOC highlights the importance of the iron-coordinating residues in the catalytic activity of EncFtn. Furthermore, the position of the calcium ion coordinated by Glu31 and Glu34 seen in the EncFtnsH structure suggests an entry site to channel metal ions into the FOC; we propose that this site binds hydrated iron ions in vivo and acts as a selectivity filter and gate for the FOC. The constellation of charged residues on the outer circumference of EncFtn (His57, Glu61 and Glu64) could function in the same way as the residues lining the mineralization surface within the classical ferritin nanocage, and given their proximity to the FOC these sites may be the exit portal and mineralization site.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>73772</offset><text>The absolute requirement for the spatial separation of oxidation and mineralization in ferritins suggests that the EncFtn family proteins are not capable of storing iron minerals due to the absence of an enclosed compartment in their structure (Figure 6—figure supplement 1B). Our biochemical characterization of EncFtn supports this hypothesis, indicating that while this protein is capable of oxidizing iron, it does not accrue mineralized iron in an analogous manner to classical ferritins. While EncFtn does not store iron itself, its association with the encapsulin nanocage suggests that mineralization occurs within the cavity of the encapsulin shell. Our ferroxidase assay data on the recombinant EncFtn-Enc nanocompartments, which accrue over 4100 iron ions per complex and form regular nanoparticles, are consistent with the encapsulin protein acting as the store for iron oxidized by the EncFtn enzyme. TEM analysis of the reaction products shows the production of homogeneous iron nanoparticles only in the EncFtn-Enc nanocompartment (Figure 8—figure supplement 1).</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>74854</offset><text>Model of iron oxidation in encapsulin nanocompartments.</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>74910</offset><text>(A) Model of EncFtnsH docking to the encapsulin shell. A single pentamer of the icosahedral T. maritima encapsulin structure (PDBID: 3DKT) is shown as a blue surface with the encapsulin localization sequence of EncFtn shown as a purple surface. The C-terminal regions of the EncFtn subunits correspond to the position of the localization sequences seen in 3DKT. Alignment of EncFtnsH with 3DKT positions the central channel directly above the pore in the 3DKT pentamer axis (shown as a grey pentagon). (B) Surface view of EncFtn within the encapsulin nanocompartment (grey and blue respectively). The lumen of the encapsulin nanocompartment is considerably larger than the interior of ferritin (shown in orange behind the encapsulin for reference) and thus allows the storage of significantly more iron. The proposed pathway for iron movement through the encapsulin shell and EncFtn FOC is shown with arrows. (C) Model ofiron oxidation within an encapsulin nanocompartment. As EncFtn is unable to mineralize iron on its surface directly, Fe2+ must pass through the encapsulin shell to access the first metal binding site within the central channel of EncFtnsH (entry site) prior to oxidation within the FOC and release as Fe3+ to the outer surface of the protein where it can be mineralized within the lumen of the encapsulin cage.</text></passage><passage><infon key="file">elife-18972-fig14.jpg</infon><infon key="id">fig14</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>76242</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.034</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>76289</offset><text>Docking the decamer structure of EncFtnsH into the pentamer of the T. maritima encapsulin Tmari_0786 (PDB ID: 3DKT) shows that the position of the C-terminal extensions of our EncFtnsH structure are consistent with the localization sequences seen bound to the encapsulin protein (Figure 14A). Thus, it appears that the EncFtn decamer is the physiological state of this protein. This arrangement positions the central ring of EncFtn directly above the pore at the five-fold symmetry axis of the encapsulin shell and highlights a potential route for the entry of iron into the encapsulin and towards the active site of EncFtn. A comparison of the encapsulin nanocompartment and the ferritin nanocage highlights the size differential between the two complexes (Figure 14B) that allows the encapsulin to store significantly more iron. The presence of five FOCs per EncFtnsH decamer and the fact that the icosahedral encapsulin nanocage can hold up to twelve of decameric EncFtn between each of the internal five-fold vertices means that they can achieve a high rate of iron mineralization across the entire nanocompartment. This arrangement of multiple reaction centers in a single protein assembly is reminiscent of classical ferritins, which has 24 FOCs distributed around the nanocage.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>77576</offset><text>Our structural data, coupled with biochemical and ICP-MS analysis, suggest a model for the activity of the encapsulin iron-megastore (Figure 14C). The crystal structure of the T. maritima encapsulin shell protein has a negatively charged pore positioned to allow the passage of Fe2+ into the encapsulin and directs the metal towards the central, negatively charged hole of the EncFtn ring (Figure 4—figure supplement 1). The five metal-binding sites on the interior of the ring (Glu31/34-sites) may select for the Fe2+ ion and direct it towards their cognate FOCs. We propose that the oxidation of Fe2+ to Fe3+ occurs within the FOC according to the model postulated by in which the FOC acts as a substrate site through which iron passes and is released on to weakly coordinating sites at the outer circumference of the protein (His57, Glu61 and Glu64), where it is able to form ferrihydrite minerals which can be safely deposited within the lumen of the encapsulin nanocompartment (Figure 14).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>78575</offset><text>Here we describe for the first time the structure and biochemistry of a new class of encapsulin-associated ferritin-like protein and demonstrate that it has an absolute requirement for compartmentalization within an encapsulin nanocage to act as an iron store. Further work on the EncFtn-Enc nanocompartment will establish the structural basis for the movement of iron through the encapsulin shell, the mechanism of iron oxidation by the EncFtn FOC and its subsequent storage in the lumen of the encapsulin nanocompartment.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>79099</offset><text>Materials and methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>79121</offset><text>Cloning</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>79129</offset><text>Genes of interest were amplified by PCR using R. rubrum ATCC 11,170 genomic DNA (DSMZ) as the template and KOD Hot Start DNA Polymerase (Novagen). Primers used in this study are listed in Supplementary file 2. PCR products were visualized in 0.8% agarose gel stained with SYBR Safe (Life Technologies, UK). Fragments of interest were purified by gel extraction (Qiagen, UK) before digestion by endonuclease restriction enzymes (Thermo Fisher Scientific, UK) at 37°C for 1 hr, followed by ligation with similarly digested vector pET-28a(+) or pACYCDuet-1 at room temperature for 1 hr. Ligation product was transformed into chemically competent Escherichia coli Top10 cells and screened against 50 ng/μl kanamycin for pET-28a(+) based constructs or 34 ng/μl chloramphenicol for pACYCDuet-1 based constructs. DNA insertion was confirmed through Sanger sequencing (Edinburgh Genomics, The University of Edinburgh, UK). Sequence verified constructs were transformed into E. coli BL21(DE3) or Tuner(DE3) for protein production. Alternatively, plasmids transformed into E. coli B834(DE3) cells were cultured in selenomethionine medium.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>80264</offset><text>Protein production and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>80300</offset><text>A single colony of E. coli BL21(DE3) or Tuner(DE3) cells, transformed with protein expression plasmid, was transferred into 10 ml LB medium, or M9 minimal medium (MM), supplemented with appropriate antibiotic, and incubated overnight at 37 °C with 200 rpm shaking. The overnight pre-culture was then inoculated into 1 liter of LB medium and incubated at 37 °C with 200 rpm shaking. Recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18°C for overnight incubation. Cells were pelleted by centrifugation at 4000 g for 20 min at 4 °C, and resuspended 10-fold (volume per gram of cell pellet) in PBS to wash cells before a second centrifugation step. Cells were resuspended in 10-times (v/w) of appropriate lysis buffer for the purification method used (see details of buffers below) and lysed by sonication on ice, with ten cycles of 30-second burst of sonication at 10 µm amplitude and 30 s of cooling. Cell lysate was clarified by centrifugation at 20,000 x g, 30 min, 4 °C; followed by filtration using a 0.22 µM syringe filter (Millipore, UK).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>81438</offset><text>Selenomethionine labelled protein was produced by growing a single colony of E. coli B834 (DE3) cells transformed with protein expression plasmids in 100 ml LB medium supplemented with appropriate antibiotic overnight at 37 °C with shaking at 200 rpm. The overnight pre-culture was pelleted by centrifugation 3,000 x g, 4 °C, 15 min and washed twice with M9 minimal medium. The washed cells were transferred to 1 liter of SeMet medium, which contains M9 minimal medium, 40 mg/L of each L-amino acid (without methionine), 40 mg/L selenomethionine, 2 mM MgSO4, 0.4% (w/v) glucose and 1 mM Fe(NH4)2(SO4)2. Cells were incubated at 37 °C with 200 rpm shaking and recombinant protein production was induced at OD600= 0.6 by the addition of 1 mM IPTG and the incubation temperature was reduced to 18 °C for overnight incubation. Cells were harvested and lysed as above.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>82309</offset><text>His-tagged protein purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>82341</offset><text>Clarified cell lysate was loaded onto a 5 ml HisTrap column (GE Healthcare, UK) pre-equilibrated with HisA buffer (50 mM Tris-HCl, 500 mM NaCl and 50 mM imidazole, pH 8.0). Unbound proteins were washed from the column with HisA buffer. His-tagged proteins were then eluted by a step gradient of 50% HisA buffer and 50% HisB buffer (50 mM Tris-HCl, 500 mM NaCl and 500 mM imidazole, pH 8.0). Fractions containing the protein of interest, as determined by 15% (w/v) acrylamide SDS-PAGE, were pooled before loading onto a gel-filtration column (HiLoad 16/600 Superdex 200, GE Healthcare) equilibrated with GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl). Fractions were subjected to 15% SDS-PAGE and those containing the protein of interest were pooled for further analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>83115</offset><text>Sucrose gradient ultracentrifugation purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>83165</offset><text>Co-expressed encapsulin and EncFtn (EncFtn-Enc) and encapsulin protein were both purified according to the protocol used by M. Sutter. Briefly, EncFtn-Enc or encapsulin was expressed based on pACYCDuet-1 vector. The E. coli cells were grown, induced, harvested and sonicated in a similar way as described above. GF buffer used in this purification contains 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl. To remove RNA contamination, the lysate was supplemented with 50 μg/ml RNase A and rotated at 10 rpm and room temperature for 2 hrs, followed by centrifugation at 34,000 x g and 4 °C for 20 min and filtering through 0.22 µM syringe filter. Proteins were pelleted through 38% (w/v) sucrose cushion by ultracentrifugation at 100,000 x g and 4 °C for 21 hrs. 10% - 50% (w/v) sucrose gradient ultracentrifugation was applied to further separate the proteins at 100,000 x g and 4 °C for 17 hrs. Protein was dialyzed against GF buffer to remove sucrose before being used in chemical assays or TEM.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>84163</offset><text>Transmission electron microscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>84196</offset><text>TEM imaging was performed on purified encapsulin, EncFtn, and EncFtn-Enc and apoferritin. Purified protein at 0.1 mg/ml concentration was spotted on glow-discharged 300 mesh carbon-coated copper grids and excess liquid wicked off with filter paper (Whatman, UK). The grids were washed with distilled water and blotted with filter paper three times before staining with 0.2% uranyl acetate, blotting and air-drying. Grids were imaged using a JEM1400 transmission electron microscope and images were collected with a Gatan CCD camera. Images were analyzed using ImageJ (NIH, Bethesda, MD) and size-distribution histograms were plotted using Prism 6 (GraphPad software). To observe iron mineral formation by TEM, protein samples at 8.5 µM concentration including EncFtnsH, encapsulin, EncFtn-Enc and apoferritin were supplemented with acidic Fe(NH4)2(SO4)2 at their maximum iron loading ratio in room temperature for 1 hr. The mixtures were subjected to TEM analysis with or without uranyl acetate staining. TEM experiments without Fe loading were repeated three times, a representative set of images are presented here. Proteins loaded with Fe and imaged by TEM were from single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>85392</offset><text>Protein crystallization and X-ray data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>85442</offset><text>EncFtnsH was purified by anion exchange and Superdex 200 size- exclusion chromatography and concentrated to 10 mg/ml (based on extinction coefficient calculation). Crystallization drops were set up using the hanging drop vapor diffusion method at 292 K. Glass coverslips were set up with 1–2 μl protein mixed with 1 μl well solution (0.14 M calcium acetate and 15% (w/v) PEG 3350) and sealed over 1 ml of well solution. Crystals appeared after 5 days and were harvested from the well using a LithoLoop (Molecular Dimensions Limited, UK), transferred briefly to a cryoprotection solution containing well solution supplemented with 1 mM FeSO4 (in 0.1% (v/v) HCl), 20% (v/v) PEG 200, and subsequently flash cooled in liquid nitrogen. Crystals of the EncFtnsHsingle mutations were produced in the same manner as for the EncFtnsH wild-type protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>86295</offset><text>All crystallographic datasets were collected on the macromolecular crystallography beamlines at Diamond Light Source (Didcot, UK) at 100 K using Pilatus 6M detectors. Diffraction data were integrated and scaled using XDS and symmetry related reflections were merged with Aimless . Data collection statistics are shown in Table 4. The resolution cut-off used for structure determination and refinement was determined based on the CC1/2 criterion proposed by.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>86754</offset><text>The structure of EncFtnsH was determined by molecular replacement using PDB ID: 3K6C as the search model, modified to match the sequence of the target protein using Chainsaw. A single solution comprising three decamers in the asymmetric unit was found by molecular replacement using Phaser. The initial model was rebuilt using Phenix.autobuild followed by cycles of refinement with Phenix.refine, with manual rebuilding and model inspection in Coot . The final model was refined with isotropic B-factors, torsional NCS restraints, and with anomalous group refinement. The model was validated using MolProbity. Structural superimpositions were calculated using Coot. Crystallographic figures were generated with PyMOL. Multiple sequence alignment of EncFtn and ferritin family proteins was performed using Clustal Omega Sievers and Higgins, 2014 and displayed with Espript 3.0. Model refinement statistics are shown in Table 4. The final models and experimental data are deposited in the PDB and diffraction image files are available at the Edinburgh DataShare repository.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>87828</offset><text>Horse spleen apoferritin preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>87865</offset><text>Horse spleen apoferritin purchased from Sigma Aldrich (UK) was dissolved in deaerated MOPS buffer (100 mM MOPS, 100 mM NaCl, 3 g/100 ml Na2S2O4 and 0.5 M EDTA, pH 6.5). Protein was dialyzed against 1 liter MOPS buffer in room temperature for two days before buffer exchanging to GF buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) in a vivaspin column with 5 kDa cut-off (Sartorius, UK) for several times. Fe content of apoferritin was detected using ferrozine assay. Protein concentration was determined using Pierce Microplate BCA Protein Assay Kit. Apoferritin containing less than 0.5 Fe per 24-mer was used in the ferroxidase assay. Apoferritin used in the Fe loading capacity experiment was prepared in the same way with 5–15 Fe per 24-mer.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>88612</offset><text>Ferroxidase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>88630</offset><text>1 mM and 200 µM Fe(NH4)2(SO4)2 stock solutions were prepared in 0.1% (v/v) HCl anaerobically. Protein solutions with 20 µM FOC were diluted from ~10 mg/ml frozen stock in GF buffer (50 mM Tris-HCl, pH 8.0 and 150 mM NaCl) anaerobically. Ferroxidase activity was initiated by adding 450 μl protein to 50 μl of acidic Fe(NH4)2(SO4)2 at the final concentration of 100 µM and 20 µM in the air, respectively. The ferroxidase activity was measured by monitoring the Fe3+ formation which gives rise to the change of the absorbance at 315 nm. Absorbance at 315 nm was recorded every second over 1800 s using a quartz cuvette in a JASCO V-730 UV/VIS spectrophotometer (JASCO Inc., Easton, MD). In recombinantly coexpressed nanocompartments the ratio of EncFtn to Enc was assumed as 2 to 1, assuming each of the twelve pentameric vertices of the icosahedral encapsulin were occupied with decameric EncFtn. The data are presented as the mean of three technical replicates with error bars indicating one standard deviation from the mean. Proteins used here were from a single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>89725</offset><text>Iron loading capacity of ferritins</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>89760</offset><text>In order to determine the maximum iron loading capacity, around 8.5 µM proteins including decameric EncFtnsH, Encapsulin, EncFtn-Enc and apoferritin were loaded with various amount of acidic Fe(NH4)2(SO4)2 ranging from 0 to 1700 µM. Protein mixtures were incubated in room temperature for 3 hrs before desalting in Zebra spin desalting columns (7 kDa cut-off, Thermo Fisher Scientific, UK) to remove free iron ions. The protein concentration was determined using PierceMicroplate BCA assay kit (Thermo Fisher Scientific). The protein standard curve was plotted according to the manufacturer. The Fe content in the samples was determined using modified ferrozine assay. Briefly speaking, 100 μl protein sample was mixed with 100 μl mixture of equal volume of 1.4 M HCl and 4.5% (w/v) KMnO4 and incubated at 60 °C for 2 hrs. 20 μl of the iron-detection reagent (6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in H2O) was added to the cooled tubes. 30 min later, 200 μl of the solution was transferred into a well of 96-well plate and the absorbance at 562 nm was measured on the plate reader Spectramax M5 (Molecular Devices, UK). The standard curve was plotted using various concentrations of FeCl3 (in 10 mM HCl) diluted in the gel-filtration buffer. Three technical repeats were performed for both the ferrozine and microplate BCA assays. Samples analyzed by ICP-MS were prepared in the same way by mixing protein and ferrous ions and desalting. The proteins used in the Fe loading experiment came from a single preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>91345</offset><text>Peroxidase assay</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>91362</offset><text>The peroxidase activity of EncFtnsH was determined by measuring the oxidation of ortho-phenylenediamine (OP) by H2O2 . EncFtnsH decameric and monomeric fractions purified from MM were both used in the assay. Ortho-phenylenediamine was prepared as a 92.5 mM stock solution in 50 mM Tris-HCl (pH 8.0). 80, 70, 60, 50, 40, 30, 20 and 10 mM of OP were prepared by diluting the stock solution in the 50 mM Tris-HCl (pH 8.0). 100 μl of each diluted OP was added to a 96-well plate in 3 repeats. 1 μl of 32 µM protein was supplemented into each well to a final concentration of 160 nM, followed by the addition of 2 μl of 30% H2O2. After 15 min shaking in the dark, the reaction was stopped by adding 100 μl of 0.5 M H2SO4. The peroxidase activity was measured by monitoring the absorbance at 490 nm in the SpectraMax M5 Microplate Reader (Molecular Devices).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>92223</offset><text>ICP-MS analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>92239</offset><text>Protein samples were diluted 50-fold into a solution of 2.5% HNO3 (Suprapur, Merck, UK) containing 20 µg/L Pt as internal standard. Matrix-matched elemental standards (containing analyte metal concentrations 0 – 1000 µg/L) were prepared by serial dilution from individual metal standard stocks (VWR) with identical solution compositions, including the internal standard. All standards and samples were analyzed by ICP-MS using a Thermo x-series instrument (Thermo Fisher Scientific) operating in collision cell mode (using 3.0 ml min-1 flow of 8% H2 in He as the collision gas). Isotopes 44Ca, 56Fe, 66Zn, 78Se, and 195Pt were monitored using the peak-jump method (100 sweeps, 25–30 ms dwell time on 5 channels per isotope, separated by 0.02 atomic mass units) in triplicate. The protein samples used in ICP-MS came from a single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>93098</offset><text>Mass spectrometry analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>93125</offset><text>For native MS analysis, all protein samples were buffer exchanged into 100 mM ammonium acetate (pH 8.0; adjusted with dropwise addition of 1% ammonia solution) using Micro Biospin Chromatography Columns (Bio-Rad, UK) prior to analysis and the resulting protein samples were analyzed at a final concentration of ~5 µM (oligomer concentration). In order to obtain Fe-bound EncFtn, 100 µM or 300 µM of freshly prepared FeCl2 was added to apo-EncFtnsH (monomer peak) immediately prior to buffer exchange into 100 mM ammonium acetate (pH 8.0). Samples were analyzed on a quadrupole ion-mobility time of flight instrument (Synapt G2, Waters Corp., Manchester, UK), equipped with a nanomate nanoelectrospray infusion robot (Advion Biosciences, Ithaca, NY). Instrument parameters were tuned to preserve non-covalent protein complexes. After optimization, typical parameters were: nanoelectrospray voltage 1.54 kV; sample cone 50 V; extractor cone 0 V; trap collision voltage 4 V; source temperature 80°C; and source backing pressure 5.5 mbar. For improved mass resolution the sample cone was raised to 155 V. Ion mobility mass spectrometry (IM-MS) was performed using the travelling-wave mobility cell in the Synapt G2, employing nitrogen as the drift gas. Typically, the IMS wave velocity was set to 300 m/s; wave height to 15 V; and the IMS pressure was 1.8 mbar. All native MS experiments were performed on samples from two independent protein preparations. For collision cross section determination, IM-MS data was calibrated using denatured equine myoglobin and data was analyzed using Driftscope v2.5 and MassLynx v4.1 (Waters Corp., UK). Theoretical collision cross sections (CCS) were calculated from pdb files using IMPACT software v. 0.9.1. In order to obtain information on the topology of the EncFtnsH assembly, gas-phase dissociation of the Fe-associated EncFtnsH complex was achieved by increasing the sample cone and/or trap collision voltage prior to MS analysis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>95109</offset><text>SEC-MALLS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>95119</offset><text>Size-exclusion chromatography (ÄKTA-Micro; GE Healthcare) coupled to UV, static light scattering and refractive index detection (Viscotec SEC-MALS 20 and Viscotec RI Detector:VE3580; Malvern Instruments, UK) were used to determine the molecular mass of fractions decamer and monomer of EncFtnsH in solution individually. Protein concentration was determined by measurement of absorbance at 280 nm and calculated using the extinction coefficient ε0.1%= 1.462 mg−1 ml-1 cm−1. 100 μl of 1.43 mgml-1 fractions of EncFtnsH decamer and 4.03 mg ml -1 fractions of EncFtnsH monomer were run individually on a Superdex 200 10/300 GL size-exclusion column pre-equilibrated in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl at 22°C with a flow rate of 0.5 ml/min. Light scattering, refractive index (RI) and A280nm were analyzed by a homo-polymer model (OmniSEC software, v 5.1; Malvern Instruments) using the following parameters for fractions of decamer and monomer: the extinction coefficient (dA/dc) at 280 nm was 1.46 AU mg ml−1 and specific refractive index increment (dn/dc) was 0.185 ml g−1. The proteins analyzed by SEC-MALLS came from single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>96289</offset><text>Metal binding analysis by PAGE</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>96320</offset><text>Recombinant EncFtnsH fractions at 50 µM concentration were incubated with one molar equivalent of metal ions at room temperature for 2 hrs. Half of each sample was mixed with 5 x native loading buffer (65 mM Tris-HCl, pH 8.5, 20% glycerol and 0.01% bromophenol blue) and run on non-denaturing PAGE gels (10% acrylamide) and run in Tris/glycine buffer, 200 V, 4 °C for 50 min. The remaining samples were left for an additional three hours prior to SDS-PAGE (15% acrylamide) analysis. SDS-PAGE gels were run at room temperature at 200 V, room temperature for 50 min. Gels were stained with Coomassie Brilliant Blue R250 and scanned after de-staining in water. The proteins used in this experiment came from single protein preparation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>97057</offset><text>Analytical size-exclusion chromatography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>97098</offset><text>For analysis of the multimeric state of EncFtn proteins by analytical size-exclusion gel-filtration chromatography (AGF) 25 μl of 90 µM protein was loaded into Superdex 200 PC 3.2/30 column (GE Healthcare) at 15 °C with GF buffer running at 0.05 ml/min and pressure limit 0.45 MPa. In order to use AGF to determine how metal ions influence the assembly of EncFtnsH, 90 µM EncFtnsH monomer fractions were mixed with equal molar concentrations of metal ion solutions including FeSO4 in 0.1% (v/v) HCl, Fe(NH4)2(SO4)2, FeCl3, CoCl2, calcium acetate (CaAc), ZnSO4 and MnCl2 at room temperature for 2 hrs prior to AGF analysis. Protein samples without metal titration were also analyzed as a control group. Both monomer and decamer fractions of EncFtnsH left at room temperature for 2 hrs, or overnight, were also analysed as controls to show the stability of the protein samples in the absence of additional metal ions. The AGF results have been repeated twice using two independent preparations of protein, of which only one representative trace is presented in the paper.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>98177</offset><text>Accession codes and datasets</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>98206</offset><text>Coordinates and structure factors for the structures presented in this paper have been deposited in the PDB under the following accession codes: EncFtnsH, 5DA5; EncFtnsH-E32A, 5L89; EncFtnsH-E62A, 5L8B; EncFtnsH-H65A, 5L8G (DOIs for X-ray diffraction image data are shown in Table 4). All MS datasets presented in this paper can be found, in the raw format at http://dx.doi.org/10.7488/ds/1449.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>98601</offset><text>Funding Information</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98621</offset><text>This paper was supported by the following grants:</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98671</offset><text> to Didi He.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98684</offset><text> to Sam Hughes, Kirsten Altenbach, David J Clarke.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98735</offset><text> to Emma Tarrant, Kevin J Waldron.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98771</offset><text> to David J Clarke, Jon Marles-Wright.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">paragraph</infon><offset>98811</offset><text> to Jon Marles-Wright.</text></passage><passage><infon key="section_type">ACK_FUND</infon><infon key="type">title_1</infon><offset>98835</offset><text>Additional information</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">title_1</infon><offset>98858</offset><text>Competing interests</text></passage><passage><infon key="section_type">COMP_INT</infon><infon key="type">footnote</infon><offset>98878</offset><text>The authors declare that no competing interests exist.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>98933</offset><text>Author contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>98954</offset><text>DH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99073</offset><text>SH, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99135</offset><text>SV-H, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99199</offset><text>AG, Acquisition of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99258</offset><text>KA, Acquisition of data, Contributed unpublished essential data or reagents.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99335</offset><text>ET, Acquisition of data, Analysis and interpretation of data.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99397</offset><text>CLM, Acquisition of data, Contributed unpublished essential data or reagents.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99475</offset><text>KJW, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99572</offset><text>DJC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">footnote</infon><offset>99692</offset><text>JM-W, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>99813</offset><text>Additional files</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_2</infon><offset>99830</offset><text>Major datasets</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>99845</offset><text>The following datasets were generated:</text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>99884</offset><text>References</text></passage><passage><infon key="fpage">276</infon><infon key="lpage">278</infon><infon key="name_0">surname:Aberg;given-names:A</infon><infon key="name_1">surname:Nordlund;given-names:P</infon><infon key="name_2">surname:Eklund;given-names:H</infon><infon key="pub-id_doi">10.1038/361276a0</infon><infon 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key="name_3">surname:Günther;given-names:S</infon><infon key="name_4">surname:Prangishvili;given-names:D</infon><infon key="name_5">surname:Loessner;given-names:MJ</infon><infon key="name_6">surname:Stetter;given-names:KO</infon><infon key="name_7">surname:Weber-Ban;given-names:E</infon><infon key="name_8">surname:Ban;given-names:N</infon><infon key="pub-id_doi">10.1038/nsmb.1473</infon><infon key="section_type">REF</infon><infon key="source">Nature Structural & Molecular Biology</infon><infon key="type">ref</infon><infon key="volume">15</infon><infon key="year">2008</infon><offset>104104</offset><text>Structural basis of enzyme encapsulation into a bacterial nanocompartment</text></passage><passage><infon key="section_type">REF</infon><infon key="type">paragraph</infon><offset>104178</offset><text>10.7554/eLife.18972.048</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>104202</offset><text>Decision letter</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104218</offset><text>Losick</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104225</offset><text>Richard</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104233</offset><text>In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104517</offset><text>[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The previous decision letter after peer review is shown below.]</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>104711</offset><text>Thank you for submitting your work entitled "Structural characterisation of an encapsulated ferritin provides insight into iron storage in bacterial nanocompartments" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105289</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105888</offset><text>Reviewer #1: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>105902</offset><text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106212</offset><text>2) Results:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106224</offset><text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106364</offset><text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>106635</offset><text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system. Thus the significance of the experimental results in the paper are unclear.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107164</offset><text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.)</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107409</offset><text>Reviewer #2: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>107423</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>108298</offset><text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>108754</offset><text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109070</offset><text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved?</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109185</offset><text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109403</offset><text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109612</offset><text>10.7554/eLife.18972.049</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">title</infon><offset>109636</offset><text>Author response</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109652</offset><text>[Editors’ note: the author responses to the first round of peer review follow.]</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>109734</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently, and thus represent an interesting frontier in microbial cell biology. While this study significantly advances our understanding of the structural and biochemical relationship between encapsulins and EncFer, it requires significant revision prior to publication. We do, however, encourage the authors to resubmit when and if they are address the issues raised below. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110334</offset><text>Reviewer #1: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110348</offset><text>1) Methods: What procedures and analyses did the author use to assess whether the iron added to the various ferritin derivatives was protein coated or was simply balls of rust attached to protein fragments? If the latter, it could easily generate reactive oxygen species in air under physiological conditions. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>110659</offset><text>The reviewer makes an excellent point here. To ascertain whether the iron in the assays forms ‘balls of rust’ we performed transmission electron microscopy on the ferroxidase reaction mixtures after completion of the reaction to assess the formation of free, or encapsulated iron minerals. We provide an additional supplemental figure (Figure 8—figure supplement 1) and discuss the observation of iron mineral crystals and nanoparticles in the main text, subsection “Ferroxidase activity”, last paragraph. We also attempted to use a commercial luminescence-based ROS detection kit on the reactions to address the possibility that H2O2 is produced as a reaction intermediate by the EncFtn protein. We found that the results from this particular kit were inconsistent between repeats, but for the benefit of the reviewer we provide a graph of the results obtained (see Author response image 1). These results show the production of ROS by apoferritin, which is consistent with the published data on the reaction mechanism of certain ferritins; however, no significant ROS were detected for the EncFtn or encapsulin proteins.</text></passage><passage><infon key="file">elife-18972-resp-fig1.jpg</infon><infon key="id">fig15</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>111792</offset><text>DOI:
http://dx.doi.org/10.7554/eLife.18972.037</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111839</offset><text>We acknowledge that the reaction mechanism of the EncFtn merits further investigation in a follow up study.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111947</offset><text>2) Results:</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>111959</offset><text>A) Critical data, such as the comparison of maximum amount of iron bound by a monomer in the dodocamer is in the Supplementary information. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112100</offset><text>We acknowledge that the data for iron loading merits inclusion in the main text, we have now moved this data and other supplementary data tables to the main text.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112263</offset><text>B) The data in Supplementary file 2, shows that the amount of iron bound by an ENCFTN decamer monomer is sub -stoichiometric, ranging from 0.18 to 0.64. In a bona fide ferritin, with ~ 2000 iron atoms/ protein cage (24 subunits), the same parameter is much, much higher. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112535</offset><text>One of the central arguments of our paper is the fact that the EncFtnsH monomer must dimerize to produce a functional ferroxidase active site and that its iron binding properties are highly divergent from those of the classical ferritin nanocages. We have added additional text to the manuscript to highlight these differences (Introduction, last paragraph, and Mass spectrometry section) and discuss the functional consequences at length.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>112975</offset><text>Even an experimental situation: 24 subunit (monomer) ferritin with a biomineral prepared experimentally from apoferritin and containing, on average, only 1000 iron atoms/24 subunit cage, the equivalent parameter appears to be 1000/24 = 42. This Fe/protein ratio is 66 times more iron than in the test system described. Moreover, in nature, some ferritin protein cages contain as much as 4500 Fe atoms, several hundred times higher than the test system! Thus the significance of the experimental results in the paper are unclear. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>113505</offset><text>We have clarified this key difference in the discussion of the iron storage function of the encapsulin nanocompartment (subsection “Iron storage in encapsulin nanocompartments”, second paragraph). The key conclusion of the paper is that the iron storage and iron oxidation functions that are combined in classical ferritins are split between the encapsulin nanocompartment and the EncFtn protein.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>113906</offset><text>3) Table 4: Data are shown for three proteins, Encapsulin, Enc-Ftn-10mer, and EncFTN-Enc. Missing are data for the starting material, 24 subunit ferritin or apoferritin (ferritin with the iron removed, by reduction and chelation, as a control.) </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114152</offset><text>The data for the starting material are shown in Table 5. Control data for apoferritin have been added to this table and are illustrated in Figure 8. We note that we do not reach the experimental maximum loading capacity for apoferritin; however, we also note that the EncFtn-encapsulin nanocompartment sequesters five times more iron than the ferritin under the same reaction conditions, supporting the published observations that these nanocompartments can store more iron than classical ferritin nanocages.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114661</offset><text>Reviewer #2: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>114675</offset><text>In this manuscript the authors characterize the iron binding and oxidation mechanisms of bacterial encapsulins. The widespread nature of these compartments and their potential physiological roles have only been appreciated recently. While the structure of the encapsulin shell has been determined, that of its cargo, the ferritin-like protein (EncFer), has remained elusive. Here, the authors provide the structure of one such cargo and show that it assembles in a manner that is topologically distinct from ferritin. Additionally, the authors provide evidence that metal binding promotes the assembly of the EncFer and that it does act as a ferroxidase. Altogether, there is a substantial amount of work here that will likely be viewed as a major step forward in understanding these unique bacterial organelles. I have a few suggestions and questions that are listed below: </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>115551</offset><text>1) The authors grow E. coli in minimal media with and without added iron to show that assembly is iron dependent. The output of these experiments is the ratio of decamer vs. monomer. However, we don't have information on whether the growth conditions altered either the total amount of protein produced or the total amount soluble complex/monomer. Perhaps, lower protein concentrations lead to less efficient assembly (a critical concentration is needed). </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>116008</offset><text>The reviewer makes an interesting point about growth conditions and we acknowledge that production of the protein in LB medium leads to varying protein yields and monomer/decamer proportions. We therefore adopted the use of M9 minimal medium throughout the study to give better reproducibility, which also enables better control of metal ion availability than the complex LB medium. Given the fact that the protein is produced recombinantly in E. coli it is not particularly instructive to prove the in vivoproduction of the EncFtn multimer in this host. We have added a panel to Figure 3 to show the effect of protein concentration on multimerization in vitro(Figure 3C). Our mass spectrometry results show that the protein spontaneously multimerized in the presence of iron in vitroto form decameric species and that this is metal ion concentration dependent (Figure 7).</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>116881</offset><text>2) There is no information regarding the reason for the use of R. rubrum encapsulins. As far as I can tell, these have not been a model for either in vivo or in vitro work. Is there even evidence that they are produced by R. rubrum? What is their size/appearance in that organism? Do they have a physiological role? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117198</offset><text>We have put a comment in the Introduction to introduce R. rubrum (last paragraph). A preliminary study in the laboratory identified encapsulins in a preparation of lipid vesicles from R. rubrum containing chromatophores. We chose to follow up on these structures in this study. We do not feel this particular information is key to the central argument of the paper.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117564</offset><text>3) Also, how similar are the Enc and EncFer to those of M. xanthus? Are the putative iron-binding sites conserved? </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117680</offset><text>We have noted this in the Introduction of the manuscript.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117738</offset><text>4) I would have liked to see some mutagenesis experiments to test the models of assembly, iron binding and ferroxidase activity. These do not have to be in vivo and can be performed in vitro with the available system. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>117957</offset><text>To address this question we have produced three FOC mutants of the EncFtn protein and characterized these in solution, by mass spectrometry, and crystallographically (section: Mutagenesis of the EncFtnsH Ferroxidase center). We thank the reviewer for this suggestion as it highlighted the importance of the FOC residues for assembly and activity, and our new data has provided interesting insights into the EncFtn protein.</text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>118380</offset><text>5) I would like some more phylogenetic data for the model that ferritin evolved from EncFer. Perhaps, EncFer evolved from ferritin? Do any of the existing phylogenetic analyses support one model over another. </text></passage><passage><infon key="section_type">REVIEW_INFO</infon><infon key="type">paragraph</infon><offset>118590</offset><text>We now include a phylogenetic tree (Figure 13) and consider the question of ferritin evolution in the Discussion (first paragraph). None of the authors of this study are evolutionary biologists but we appreciate the difficulty inherent in tracing the history of protein folds, especially in bacterial lineages. We refer to previous studies in this section and make a suggestion that can be followed up in subsequent studies.</text></passage></document></collection>
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<collection><source>PMC</source><date>20201220</date><key>pmc.key</key><document><id>5014086</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1016/j.str.2016.06.020</infon><infon key="article-id_pmc">5014086</infon><infon key="article-id_pmid">27524201</infon><infon key="article-id_publisher-id">S0969-2126(16)30167-8</infon><infon key="fpage">1599</infon><infon key="issue">9</infon><infon key="license">This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</infon><infon key="lpage">1605</infon><infon key="name_0">surname:Zebisch;given-names:Matthias</infon><infon key="name_1">surname:Jackson;given-names:Verity A.</infon><infon key="name_2">surname:Zhao;given-names:Yuguang</infon><infon key="name_3">surname:Jones;given-names:E. Yvonne</infon><infon key="notes">Published: August 11, 2016</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">24</infon><infon key="year">2016</infon><offset>0</offset><text>Structure of the Dual-Mode Wnt Regulator Kremen1 and Insight into Ternary Complex Formation with LRP6 and Dickkopf</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>115</offset><text>Summary</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>123</offset><text>Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between β-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1127</offset><text>Graphical Abstract</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract_title_1</infon><offset>1146</offset><text>Highlights</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1157</offset><text>The structure of the KREMEN 1 ectodomain is solved from three crystal forms</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1233</offset><text>Kringle, WSC, and CUB subdomains interact tightly to form a single structural unit</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1316</offset><text>The interface to DKKs is formed from the Kringle and WSC domains</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1381</offset><text>The CUB domain is found to interact directly with LRP6PE1PE2</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1442</offset><text>Zebisch et al. describe the ectodomain structure of KREMEN 1, a receptor for Wnt antagonists of the DKK family. Apo structures and a complex with functional fragments of DKK1 and LRP6 shed light on the function of this dual-mode regulator of Wnt signaling.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1700</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1713</offset><text>Signaling by Wnt morphogens is renowned for its fundamental roles in embryonic development, tissue homeostasis, and stem cell maintenance. Due to these functions, generation, delivery, and interpretation of Wnt signals are all heavily regulated in the animal body. Vertebrate Dickkopf proteins (Dkk1, 2, and 4) are one of many secreted antagonists of Wnt and function by blocking access to the Wnt co-receptor LRP5/6. Kremen proteins (Krm1 and Krm2) have been identified as additional high-affinity transmembrane receptors for Dkk. Krm and Dkk synergize in Wnt inhibition during Xenopus embryogenesis to regulate anterior-posterior patterning. Mechanistically it is thought that, in the presence of Dkk, Krm forms a ternary complex with Lrp6, which is then rapidly endocytosed. This amplifies the intrinsic Wnt antagonistic activity of Dkk by efficiently depleting the cell surface of the Wnt co-receptor. In accordance with this, Krm1−/− and Krm2−/− double knockout mice show a high bone mass phenotype typical of increased Wnt signaling, as well as growth of ectopic forelimb digits. Growth of ectopic digits is further enhanced upon additional loss of dkk expression. The Wnt antagonistic activity of Krm1 is also linked to its importance for correct thymus epithelium formation in mice. The importance of intact KRM1 for normal human development and health is highlighted by the recent finding that a homozygous mutation in the ectodomain of KRM1 leads to severe ectodermal dysplasia including oligodontia. Interestingly, the Wnt antagonistic activity of Krm is context dependent, and Krm proteins are actually dual-mode Wnt regulators. In the absence of Dkk, Krm1 and 2 change their function from inhibition to enhancement of Lrp6-mediated signaling. By direct binding to Lrp6 via the ectodomains, Krm proteins promote Lrp6 cell-surface localization and hence increase receptor availability. Further increasing the complexity of Krm functionality, it was recently found that Krm1 (but not Krm2) can also act independently of LRP5/6 and Wnt as a dependence receptor, triggering apoptosis unless bound to Dkk.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3834</offset><text>Structurally, Krm1 and 2 are type I transmembrane proteins with a 40 kDa ectodomain and a flexible cytoplasmic tail consisting of 60–75 residues. The ectodomain consists of three similarly sized structural domains of around 10 kDa each: the N-terminal Kringle domain (KR) is followed by a WSC domain of unknown fold. The third structural domain is a CUB domain. An approximately 70-residue linker connects the CUB domain to the transmembrane span. An intact KR-WSC-CUB domain triplet and membrane attachment is required for Wnt antagonism. The transmembrane span and cytoplasmic tail can be replaced with a GPI linker without impact on Wnt antagonism.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4490</offset><text>We sought to provide structural insights into the multi-functionality of this cell-surface receptor. The structures presented here reveal the unknown fold of the WSC domain and the tight interactions of all three domains. We further succeeded in determination of a low-resolution LRP6PE3PE4-DKK1CRD2-KRM1ECD complex, defining the architecture of the Wnt inhibitory complex that leads to Lrp6 cell-surface depletion.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>4907</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>4915</offset><text>The recombinant production of the extracellular domain of Krm for structural studies proved challenging (see Experimental Procedures). We succeeded in purifying KRM1ECD complexes with DKK1fl, DKK1Linker-CRD2, and DKK1CRD2 that were monodisperse and stable in gel filtration, hence indicating at least micromolar affinity (data not shown). Several crystal forms were obtained from these complexes, however, crystals always contained only KRM1 protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5366</offset><text>We solved the structure of KRM1ECD in three crystal forms at 1.9, 2.8, and 3.2 Å resolution (Table 1). The high-resolution structure is a near full-length model (Figure 1). The small, flexible, and charged 98AEHED102 loop could only be modeled in a slightly lower resolution structure and in crystal form III. The KR, WSC, and CUB are arranged in a roughly triangular fashion with tight interactions between all three domains. The KR domain, which bears two of the four glycosylation sites, contains the canonical three disulfide bridges (C32-C114, C55-C95, C84-C109) and, like other Kringle domains, is low in secondary structure elements. The structurally most similar Kringle domain is that of human plasminogen (PDB: 1PKR) with an root-mean-square deviation (RMSD) of 1.7 Å for 73 aligned Cα (Figure 1B). The KRM1 structure reveals the fold of the WSC domain for the first time. The structure is best described as a sandwich of a β1-β5-β3-β4-β2 antiparallel β sheet and a single α helix. The structure is also rich in loops and is stabilized by four disulfide bridges (C122-C186, C147-C167, C151-C169, C190-C198). Using the PDBeFold server, we detected a surprising yet significant homology to PAN module domains. The closest structural relative is hepatocyte growth factor (HGF, PDB: 1GP9), which superposes with an RMSD of 2.3 Å for 58 aligned Cα (Figure 1B). The CUB domain bears two glycosylation sites. Although present, the quality of the electron density around N217 did not allow modeling of the sugar moiety. In crystal form I, a calcium ion is present at the canonical position coordinated by the carboxylates of D263, D266 (bidentate), and D306, as well as the carbonyl of N309 and a water molecule. The coordination sphere deviates significantly from perfectly octahedral (not shown). This might result in the site having a low affinity and may explain why calcium is not present in the two low-resolution crystal forms. Loss of calcium has led to loop rearrangements and partial disorder in these crystal forms. The closest structural relative is the CUB_C domain of Tsg-6 (PDB: 2WNO), which superposes with KRMCUB with an RMSD of 1.6 Å for 104 Cα (Figure 1B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7578</offset><text>A superposition of the three KRM1 structures reveals no major structural differences (Figure 1C) as anticipated from the plethora of interactions between the three domains. Minor differences are caused by the collapse of the Ca2+ binding site in crystal forms II and III and loop flexibility in the KR domain. The F207S mutation recently found to cause ectodermal dysplasia in Palestinian families maps to the hydrophobic core of the protein at the interface of the three subdomains (Figure 1A). Such a mutation is bound to severely destabilize the protein structure of KRM1, leading to disturbance of its Wnt antagonistic, Wnt stimulatory, and Wnt independent activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8251</offset><text>Low-Resolution Insight into Ternary Complex Formation</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8305</offset><text>Co-crystallization of LRP6PE3PE4 with DKK1CRD2, and LRP6PE1 with an N-terminal peptide of DKK1 has provided valuable structural insight into direct Wnt inhibition by Dkk ligands. One face of the rather flat DKK1CRD2 fragment binds to the third β propeller of LRP6. Mutational analyses further implied that the LRP6PE3-averted face of DKK1CRD2 bears the Krm binding site, hence suggesting how Dkk can recruit both receptors into a ternary complex.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8755</offset><text>To obtain direct insight into ternary complex formation by Lrp5/6, Dkk, and Krm, we subjected an LRP6PE3PE4-DKK1fl-KRM1ECD complex to crystallization trials. Diffraction data collected from the resulting crystals were highly anisotropic with diffraction extending in the best directions to 3.5 Å and 3.7 Å but only to 6.4 Å in the third direction. Despite the lack of high-resolution diffraction, the general architecture of the ternary complex is revealed (Figure 2A). DKK1CRD2 binds to the top face of the LRP6 PE3 β propeller as described earlier for the binary complex. KRM1ECD does indeed bind on the opposite side of DKK1CRD2 with only its KR and WSC domains engaged in binding (Figure 2A). Although present in the complex subjected to crystallization, we observe no density that could correspond to CRD1 or the domain linker (L). We confirm that the CRD2 of DKK1 is required and sufficient for binding to KRM1: In surface plasmon resonance (SPR), we measured low micromolar affinity between full-length DKK1 and immobilized KRM1ECD (Figure 2B). A SUMO fusion of DKK1L-CRD2 displayed a similar (slightly higher) affinity. In contrast, a SUMO fusion of DKK1CRD1-L did not display binding for concentrations tested up to 325 μM (Figure 2B).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10014</offset><text>Overall, the DKK1-KRM1 interface is characterized by a large number of polar interactions but only few hydrophobic contacts (Figure 2C). The crystal structure gives an explanation for DKK1 loss-of-binding mutations identified previously: R191 of DKK1 forms a double salt bridge to D125 and E162 of KRM1 (Figure 2C). A charge reversal as in the mouse Dkk1 (mDkk1) R197E variant would severely disrupt the binding. Similarly, the K226 side chain of DKK1, which points to a small hydrophobic pocket on the surface of KRM1 formed by Y108, W94, and W106, forms salt bridges with the side chains of KRM1 D88 and D90. Again, a charge reversal as shown before for mDkk1 K232E would be incompatible with binding. The side chain of DKK1 S192 was also predicted to be involved in Krm binding. Indeed, we found (Figure 2C) that the side chain of D201 of KRM1 forms two hydrogen bonds to the side-chain hydroxyl and the backbone amide of S192 (mouse, S198). Additional polar interactions are formed between the N140, S163, and Y165 side chains of KRM1 and DKK1 backbone carbonyls of W206, L190, and C189, respectively. The carbonyl of DKK1 R224 is hydrogen bonded to Y105 and W106 of KRM1. We suspect that the Dkk charge reversal mutations performed in the murine background and shown to diminish Krm binding K211E and R203E (mouse K217E and R209E) do so likely indirectly by disruption of the Dkk fold. We further validated the DKK1 binding site on KRM1 by introducing glycosylation sites at the KR (90DVS92→NVS) and WSC (189VCF191→NCS) domains pointing toward DKK (Figures 2A and 2D). Introduction of N-linked glycans in protein-protein-binding sites is an established way of disrupting protein-binding interfaces. Both ectodomain mutants were secreted comparably with the wild-type, indicating correct folding, but failed to achieve any detectable binding in SPR using full-length DKK1 as analyte. In contrast, a mutant carrying an additional N-glycan outside the interface at the CUB domain (309NQA311→NQS), was wild-type-like in DKK1 binding (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12076</offset><text>Identification of a Direct LRP6-KRM1 Binding Site</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12126</offset><text>The LRP6PE3PE4-DKK1CRD2-KRM1ECD complex structure reveals no direct interactions between KRM1 and LRP6. We constructed in silico a ternary complex with a close to full-length LRP6 ectodomain (PE1PE2PE3PE4 horse shoe) similar to but without refinement against electron microscopy (EM) or small-angle X-ray scattering data. An auxiliary PE3PE4 fragment was superimposed via PE4 onto PE3 of the crystal structure, and the LRP6PE1PE2 structure was superimposed via PE2 onto PE3 of this auxiliary fragment (Figure 3A).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12642</offset><text>For this crude approximation of a true ternary complex, we noted very close proximity between the Ca2+-binding region of KRM1 and the top face of the PE2 β propeller of LRP6. The solvent-exposed residues R307, I308, and N309 of the central Ca2+-binding β connection loop of KRM1 would be almost ideally positioned for binding to this face, which is commonly used as a binding site on β propellers. Peptides containing arginine/lysine, isoleucine, and asparagine (consensus sequence N-X-I-(G)-R/K) are also employed by DKK1 and SOST to bind to LRP6 (albeit to propeller 1; Figure 3B). To support the hypothesis that KRM1CUB binds to LRP6PE2, we used SPR and compared binding of the wild-type and the GlycoCUB mutant of KRM1ECD (bearing an N-glycosylation site at N309) with a purified LRP6PE1PE2 fragment. Indeed, we found that in the absence of Dkk, KRM1ECD bound with considerable affinity to LRP6PE1PE2 (Figure 3C). In contrast, no saturable binding was observed between KRM1 and LRP6PE3PE4. Introduction of an N-glycosylation site at N309 in KRM1ECD abolished LRP6PE1PE2 binding (Figure 3C), while binding to DKK1 was unaffected (Figure 2D). We conclude that the predicted binding site between KRM1CUB and LRP6PE2 is a strong candidate for mediating the direct Lrp6-Krm interaction, which is thought to increase Wnt responsiveness by stabilizing Lrp6 at the cell surface. Further experiments are required to pinpoint the exact binding site. Although LRP6PE1 appears somewhat out of reach in the modeled ternary complex, it cannot be excluded as the Krm binding site in the ternary complex and LRP6-Krm binary complex. The presence of DKK may govern which propeller (PE1 versus PE2) of LRP6 is available for Krm binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14374</offset><text>Apparent binding across the proposed KRM1CUB-LRP6PE2 interface is expected to be higher once Krm is also cross-linked to LRP6PE3 via DKK1CRD2 (Figure 3D). Low-resolution negative-stain EM and small-angle X-ray scattering studies of LRP6PE1PE2PE3PE4, in isolation and in complex with Dkk1, plus negative-stain EM of full-length LRP6 ectodomain, have indicated curved, platform-like conformations but also potential flexibility between PE2 and PE3. It is therefore possible that the interplay of Krm and Dkk binding can promote changes in LRP6 ectodomain conformation with functional consequences; however, such ideas await investigation.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15012</offset><text>Taken together, the structural and biophysical studies we report here extend our mechanistic understanding of Wnt signal regulation. We describe the ectodomain structure of the dual Wnt regulator Krm1, providing an explanation for the detrimental effect on health and development of a homozygous KRM1 mutation. We also reveal the interaction mode of Krm-Dkk and the architecture of the ternary complex formed by Lrp5/6, Dkk, and Krm. Furthermore, the ternary crystal structure has guided in silico and biophysical analyses to suggest a direct LRP6-KRM1 interaction site. Our findings provide a solid foundation for additional studies to probe how ternary complex formation triggers internalization, whereas Krm binding in the absence of Dkk stabilizes the Wnt co-receptor at the cell surface.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>15806</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>15830</offset><text>Large-Scale Mammalian Expression and Protein Purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>15888</offset><text>KrmECD fragments were cloned into pHLsec or variants thereof. Full ectodomain variants (e.g., KRM1 isoform 3, P30-T377) were well secreted into the conditioned medium (CM) of HEK293T cells, but exhibited extensive O-glycosylation (as judged from smeary bands in western blot), which would be detrimental to crystallization. Fragments truncated to the KR-WSC-CUB core gave sharp bands but were barely secreted. We therefore engineered an A23-G373 (isoform 1 numbering used throughout the article) full ectodomain construct (KRM1ECD-TEV) with a C-terminal His10 tag that contained a TEV protease cleavage site after E324. The expected sequence of the secreted protein is ETG-23APSPGLGPGPE31 … 320AVKEE324-GSENLYFQGGS-325LPQ … VPG373-THHHHHHHHHH (the isoform-2-specific PG insertion and the TEV site are underlined). This construct was well secreted and could be processed using TEV protease. However, 80%–90% of the protein eluted as aggregates from a size-exclusion column even before TEV treatment. The same applied to analog constructs for Krm1 from zebrafish, frog, and mouse. No monomeric protein at all could be obtained for several Krm2 constructs from multiple species. A KRM1ECD-TEV expressing stable GntI-deficient HEK293S cell line was generated by excision of an EcoRI-XhoI fragment, sub-cloning into pNeo-Sec-1, and selection of neomycin-resistant cells. The stable cell line showed expression levels superior to transiently transfected cells (not shown).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17365</offset><text>Human LRP6PE1PE2, LRP6PE3PE4, and full-length DKK1 were produced in a similar way as described. Shorter constructs of DKK1 lacking the N-terminal flexible region and CRD1 were not secreted from HEK cells. However, using the approach of an N-terminal fusion to a modified SUMO protein as described earlier, we succeeded in secretory expression of a SUMO-DKK1Linker-CRD2 construct encompassing residues S141-H266. A variant of this containing a TEV cleavage site just before T181, SUMO-DKK1Linker-TEV-CRD2, was also well expressed and allowed removal of the flexible linker region.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>17945</offset><text>To obtain complexes of KRM1ECD-TEV, we (co-)transfected the stable cell line with DKK and LRP6PE3PE4 constructs described earlier. Binary and ternary KRM1ECD-DKK1fl and KRM1ECD-DKK1fl-LRP6PE3PE4 complexes were stable in gel-filtration eluting as distinct monodisperse peaks.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>18220</offset><text>Crystallization and Data Collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>18256</offset><text>All samples subjected to crystallization were purified from CM by affinity and size-exclusion chromatography. After treatment with TEV protease and endoglycosidase F1 overnight using mass equivalents of 1%, samples were subjected to size-exclusion chromatography in 10 mM HEPES/NaOH (pH 7.5), 150 mM NaCl. The crystals giving rise to the 1.9 Å dataset for KRM1 in crystal form I were obtained from a KRM1ECD-DKK1Linker-CRD2 complex concentrated to 12 mg/mL. Out of this complex, KRM1ECD crystallized alone in 2.0 M ammonium sulfate, 5% (v/v) iso-propanol. For cryoprotection, crystals were transferred to mother liquor mixed 1:1 with 3.4 M sodium malonate (pH 7.0). The slightly less well-ordered crystal of crystal form I and crystals of form II were obtained from a KRM1ECD-DKK1CRD2 complex using the SUMO-DKK1Linker-TEV-CRD2 construct and releasing SUMO and the DKK linker region by TEV and 3C protease treatment. Crystals of form I (2.1 Å) appeared from protein at 12 mg/mL in 1.0 M (NH4)H2PO4, 0.100 M sodium citrate (pH 5.6) and were cryoprotected by transfer to 2.9 M sodium malonate (pH 5.0). Crystals of form II grew from protein concentrated to 17 mg/mL in 1.0 M MgSO4, 0.1 M trisodium citrate (final pH 5.6). For cryoprotection, crystals were transferred to mother liquor mixed 1:3 with 3.0 M ammonium sulfate, 18% glycerol. Crystal form III appeared after 11 months in a dried-out drop of condition H5 of the Morpheus screen. The protein concentration had been 9 mg/mL. For cryoprotection, fresh liquid from Morpheus/H5 was added. The ternary complex structure was obtained from an LRP6PE3PE4-DKK1fl-KRM1ECD complex at 9 mg/mL that grew in condition E10 of the PACTpremier screen (pH approximately 6.8) over the course of 2–11 months. For cryoprotection, 10% PEG200 was added. By mistake, the crystals were incubated for 1 hr with 1 mM platinum compound in this cryosolution before cryocooling.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>20188</offset><text>Structure Determination</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20212</offset><text>Diffraction data were collected at DIAMOND synchrotron light source at the beamlines detailed in Table 1. The structure was initially solved from crystal form III by molecular replacement (MR) with PHASER, placing models for the CUB domain (PDB: 2WNO, CUB_C domain of Tsg-6, 37% sequence identity), and the KR domain (PDB: 1PKR, Kringle 1 of plasminogen; 39% sequence identity). Traceable density for the WSC domain became immediately evident. The KRM1 structure was then built and refined by cycling between the various crystal forms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>20748</offset><text>For the ternary complex, we obtained only a low-resolution, highly anisotropic dataset extending to Bragg spacings of 3.5 Å, 6.4 Å, and 3.7 Å along the three principle directions (<I/σI> = 2). All data to 3.5 Å were used during structure determination by MR. LRP6PE3PE4 (PDB: 4A0P) and KRM1ECD (both stripped of glycosylation sites) could be placed independently by PHASER, giving Z scores of >10 and log likelihood gains (LLG) of >200. The combined LLG was 673, increasing to 901 after rigid-body refinement. Strong electron density became apparent at glycosylation sites and close to methionines (see platinum soak above), further supporting the MR solution. Additional strong density was evident between LRP6 and KRM1, suggesting the presence of DKK1. A model of the DKK1CRD2 (PDB: 3S2K and 3S8V) could then be placed with PHASER by testing all rotation function peaks. This increased the LLG from 901 to 973 indicating a correct solution. The individually placed LRP6 and DKK models were then replaced with chains B and C from the LRP6-DKK complex in PDB: 3S2K. The structure was subjected to rigid-body refinement using single structural domains as individually positioned bodies.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>21945</offset><text>We then performed restrained refinement of the coordinates against the ellipsoidally truncated and anisotropically scaled diffraction data as obtained from the diffraction anisotropy server at UCLA. The resolution cutoffs were 3.5 Å, 6.4 Å, and 3.7 Å. Strong geometric restraints generated by PROSMART from the available high-resolution reference structures were used during refinement. No manual model building was attempted. Restrained refinement was followed by ten cycles of structure idealization. The final model had Rwork/Rfree errors of 32.5%/36.1% against the anisotropy-corrected data and 32.1%/35.5% against the unmodified but ellipsoidally truncated diffraction data.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>22631</offset><text>Surface Plasmon Resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>22657</offset><text>Equilibrium experiments were performed as described before with the addition of 2 mM CaCl2 for experiments investigating the direct LRP6PE1PE2-KRM1ECD interaction.</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>22822</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>22843</offset><text>M.Z. and V.A.J. performed experiments with support from Y.Z., who generated the stable cell line. 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Cysteines as ball and sticks, glycosylation sites as sticks. The bound calcium is shown as a gray sphere. The site of the F207S mutation associated with ectodermal dysplasia in humans is shown as mesh.</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27356</offset><text>(B) Superposition of the three KRM1ECD subdomains (solid) with their next structurally characterized homologs (half transparent).</text></passage><passage><infon key="file">gr1.jpg</infon><infon key="id">fig1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27486</offset><text>(C) Superposition of KRM1ECD from the three crystal forms. Alignment scores for each pairing are indicated on the dashed triangle.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27617</offset><text>Insight into Ternary Complex Formation</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27656</offset><text>(A) The structure of the ternary LRP6PE3PE4-DKK1CRD2-KRM1ECD complex. DKK1 (orange) is sandwiched between the PE3 module of LRP6 (blue) and the KR-WSC domain pair of KRM1 (green). Colored symbols indicate introduced N-glycan attachment sites (see D).</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>27907</offset><text>(B) SPR data comparing binding of full-length DKK1 and SUMO fusions of DKK1 truncations for binding to immobilized wild-type KRM1ECD.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28041</offset><text>(C) Close-up view of the DKK1CRD2-KRM1ECD interface. Residues involved in interface formation are shown as sticks; those mentioned in the text are labeled. Salt bridges are in pink and hydrogen bonds in black. Model bias cannot be excluded as single atoms and bonds are not resolved at 6.4–3.5 Å. See also Figure S1.</text></passage><passage><infon key="file">gr2.jpg</infon><infon key="id">fig2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28364</offset><text>(D) SPR binding data comparing DKK1 analyte binding with wild-type KRM1ECD and three variants bearing engineered glycosylation sites on the KR and WSC domains (green and blue pointing to DKK1) and on the CUB domain (orange). See also symbols in (A).</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28614</offset><text>LRP6-KRM1 Direct Interaction and Summary</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28655</offset><text>(A) In a construction of a ternary complex with all four β propellers of LRP6 intact, the CUB domain points via its Ca2+-binding region toward the top face of the second β propeller.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>28844</offset><text>(B) Close-up view of the potential interaction site. In addition, LRP6PE2 has been superimposed with DKK1 (yellow) and SOST (pink) peptide complexes of LRP6PE1.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29005</offset><text>(C) SPR measurements comparing LRP6PE1PE2 binding with wild-type KRM1ECD and the GlycoCUB mutant bearing an N-glycan at N309.</text></passage><passage><infon key="file">gr3.jpg</infon><infon key="id">fig3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>29131</offset><text>(D) Schematic representation of structural and biophysical findings and their implications for Wnt-dependent (left, middle) and independent (right) signaling. Conformational differences in the depictions of LRP6 are included purely for ease of representation.</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>29391</offset><text>Diffraction and Refinement Statistics</text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead><tr><th/><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>KRM1<sub>ECD</sub></th><th>LRP6<sub>PE3PE4</sub>-DKK<sub>CRD2</sub>-KRM1<sub>ECD</sub></th></tr></thead><tbody><tr><td>Crystal form</td><td>I</td><td>I</td><td>II</td><td>III</td><td>I</td></tr><tr><td>X-ray source</td><td>Diamond i04</td><td>Diamond i03</td><td>Diamond i03</td><td>Diamond i04</td><td>Diamond i04</td></tr><tr><td>Wavelength (Å)</td><td>0.9793</td><td>0.9700</td><td>0.9700</td><td>0.9795</td><td>0.9795</td></tr><tr><td>Space group</td><td><italic>P</italic>3<sub>1</sub>21</td><td><italic>P</italic>3<sub>1</sub>21</td><td><italic>P</italic>4<sub>3</sub></td><td><italic>P</italic>4<sub>1</sub>2<sub>1</sub>2</td><td><italic>C</italic>222<sub>1</sub></td></tr><tr><td>Unit cell a/α (Å/°)</td><td>50.9/90</td><td>50.5/90</td><td>65.8/90</td><td>67.8/90</td><td>86.9/90</td></tr><tr><td>b/β (Å/°)</td><td>50.9/90</td><td>50.5/90</td><td>65.8/90</td><td>67.8/90</td><td>100.1/90</td></tr><tr><td>c/γ (Å/°)</td><td>188.4/120</td><td>187.4/120</td><td>75.0/90</td><td>198.2/90</td><td>270.7/90</td></tr><tr><td>Wilson B factor (Å<sup>2</sup>)</td><td>31</td><td>41</td><td>76</td><td>77</td><td>NA</td></tr><tr><td>Resolution range (Å)</td><td>47.10–1.90 (1.95–1.90)</td><td>62.47–2.10 (2.16–2.10)</td><td>75.00–2.80 (2.99–2.80)</td><td>67.80–3.20 (3.42–3.20)</td><td>67.68–3.50 (7.16–6.40, 3.92–3.50)</td></tr><tr><td>Unique reflections</td><td>23,300 (1,524)</td><td>17,089 (1,428)</td><td>7,964 (1,448)</td><td>8,171 (1,343)</td><td>8,070 (723, 645)</td></tr><tr><td>Average multiplicity</td><td>9.1 (9.2)</td><td>5.2 (5.3)</td><td>3.7 (3.7)</td><td>22.7 (12.6)</td><td>3.8 (3.5, 4.4)</td></tr><tr><td>Completeness (%)</td><td>99.8 (98.5)</td><td>100 (100)</td><td>99.8 (100)</td><td>98.8 (93.4)</td><td>51.6 (98.5, 14.1)</td></tr><tr><td>&lt;<italic>I</italic>/<italic>σI</italic>&gt;</td><td>11.4 (1.7)</td><td>12.0 (1.7)</td><td>14.9 (1.5)</td><td>13.1 (1.9)</td><td>4.6 (4.1, 2.2)</td></tr><tr><td><italic>R</italic><sub>merge</sub> (%)</td><td>14.8 (158.3)</td><td>9.3 (98.0)</td><td>6.2 (98.9)</td><td>29.8 (142.2)</td><td>44.9 (40.5, 114.2)</td></tr><tr><td><italic>R</italic><sub>pim</sub> (%)</td><td>15.7 (55.3)</td><td>10.3 (109.0)</td><td>3.7 (53.8)</td><td>6.3 (40.0)</td><td>24.7 (23.9, 59.9)</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Refinement</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td><italic>R</italic><sub>work</sub> (%)</td><td>17.9</td><td>18.4</td><td>21.6</td><td>20.2</td><td>32.1</td></tr><tr><td><italic>R</italic><sub>free</sub> (%)</td><td>22.7</td><td>23.2</td><td>30.7</td><td>27.1</td><td>35.5</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>No. of Non-Hydrogen Atoms</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Protein</td><td>2,260</td><td>2,301</td><td>2,102</td><td>2,305</td><td>7,730</td></tr><tr><td>N-glycans</td><td>42</td><td>42</td><td>28</td><td>28</td><td>0</td></tr><tr><td>Water</td><td>79</td><td>54</td><td>0</td><td>2</td><td>0</td></tr><tr><td>Ligands</td><td>6</td><td>6</td><td>2</td><td>5</td><td>0</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Average B factor (Å<sup>2</sup>)</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Protein</td><td>63</td><td>65</td><td>108</td><td>84</td><td>–</td></tr><tr><td>N-glycans</td><td>35</td><td>46</td><td>102</td><td>18</td><td>–</td></tr><tr><td>Water</td><td>68</td><td>85</td><td>–</td><td>75</td><td>–</td></tr><tr><td>Ligands</td><td>36</td><td>47</td><td>91</td><td>75</td><td>66</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>RMSD from Ideality</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Bond lengths (Å)</td><td>0.020</td><td>0.016</td><td>0.019</td><td>0.016</td><td>0.004</td></tr><tr><td>Bond angles (°)</td><td>2.050</td><td>1.748</td><td>1.952</td><td>1.796</td><td>0.770</td></tr><tr><td colspan="6"><hr/></td></tr><tr><td colspan="6"><bold>Ramachandran Plot</bold></td></tr><tr><td colspan="6"><hr/></td></tr><tr><td>Favored (%)</td><td>96.8</td><td>95.5</td><td>96.9</td><td>94.9</td><td>92.3</td></tr><tr><td>Allowed (%)</td><td>99.7</td><td>100.0</td><td>100.0</td><td>99.7</td><td>99.8</td></tr><tr><td>Number of outliers</td><td>1</td><td>0</td><td>0</td><td>1</td><td>2</td></tr><tr><td>PDB code</td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWS" id="intref0085">5FWS</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWT" id="intref0090">5FWT</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWU" id="intref0095">5FWU</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWV" id="intref0100">5FWV</ext-link></td><td><ext-link ext-link-type="uri" xlink:href="pdb:5FWW" id="intref0105">5FWW</ext-link></td></tr></tbody></table>
</infon><offset>29429</offset><text> KRM1ECD KRM1ECD KRM1ECD KRM1ECD LRP6PE3PE4-DKKCRD2-KRM1ECD Crystal form I I II III I X-ray source Diamond i04 Diamond i03 Diamond i03 Diamond i04 Diamond i04 Wavelength (Å) 0.9793 0.9700 0.9700 0.9795 0.9795 Space group P3121 P3121 P43 P41212 C2221 Unit cell a/α (Å/°) 50.9/90 50.5/90 65.8/90 67.8/90 86.9/90 b/β (Å/°) 50.9/90 50.5/90 65.8/90 67.8/90 100.1/90 c/γ (Å/°) 188.4/120 187.4/120 75.0/90 198.2/90 270.7/90 Wilson B factor (Å2) 31 41 76 77 NA Resolution range (Å) 47.10–1.90 (1.95–1.90) 62.47–2.10 (2.16–2.10) 75.00–2.80 (2.99–2.80) 67.80–3.20 (3.42–3.20) 67.68–3.50 (7.16–6.40, 3.92–3.50) Unique reflections 23,300 (1,524) 17,089 (1,428) 7,964 (1,448) 8,171 (1,343) 8,070 (723, 645) Average multiplicity 9.1 (9.2) 5.2 (5.3) 3.7 (3.7) 22.7 (12.6) 3.8 (3.5, 4.4) Completeness (%) 99.8 (98.5) 100 (100) 99.8 (100) 98.8 (93.4) 51.6 (98.5, 14.1) <I/σI> 11.4 (1.7) 12.0 (1.7) 14.9 (1.5) 13.1 (1.9) 4.6 (4.1, 2.2) Rmerge (%) 14.8 (158.3) 9.3 (98.0) 6.2 (98.9) 29.8 (142.2) 44.9 (40.5, 114.2) Rpim (%) 15.7 (55.3) 10.3 (109.0) 3.7 (53.8) 6.3 (40.0) 24.7 (23.9, 59.9) Refinement Rwork (%) 17.9 18.4 21.6 20.2 32.1 Rfree (%) 22.7 23.2 30.7 27.1 35.5 No. of Non-Hydrogen Atoms Protein 2,260 2,301 2,102 2,305 7,730 N-glycans 42 42 28 28 0 Water 79 54 0 2 0 Ligands 6 6 2 5 0 Average B factor (Å2) Protein 63 65 108 84 – N-glycans 35 46 102 18 – Water 68 85 – 75 – Ligands 36 47 91 75 66 RMSD from Ideality Bond lengths (Å) 0.020 0.016 0.019 0.016 0.004 Bond angles (°) 2.050 1.748 1.952 1.796 0.770 Ramachandran Plot Favored (%) 96.8 95.5 96.9 94.9 92.3 Allowed (%) 99.7 100.0 100.0 99.7 99.8 Number of outliers 1 0 0 1 2 PDB code 5FWS 5FWT 5FWU 5FWV 5FWW </text></passage><passage><infon key="file">tbl1.xml</infon><infon key="id">tbl1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>31236</offset><text>Values in parentheses refer to the highest-resolution shell. An additional shell given for the ternary complex corresponds to the last shell with near-complete diffraction data. NA, not announced.</text></passage></document></collection>
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<collection><source>PMC</source><date>20230813</date><key>pmc.key</key><document><id>5063996</id><infon key="license">CC BY</infon><passage><infon key="alt-title">Mechanism of Arabinoxylanase</infon><infon key="article-id_doi">10.1074/jbc.M116.743948</infon><infon key="article-id_pmc">5063996</infon><infon key="article-id_pmid">27531750</infon><infon key="article-id_publisher-id">M116.743948</infon><infon key="fpage">22149</infon><infon key="issue">42</infon><infon key="kwd">cellulosome crystallography enzyme kinetics enzyme mechanism glycoside hydrolase</infon><infon key="license">Author's Choice—Final version free via Creative Commons CC-BY license.</infon><infon key="lpage">22159</infon><infon key="name_0">surname:Labourel;given-names:Aurore</infon><infon key="name_1">surname:Crouch;given-names:Lucy I.</infon><infon key="name_10">surname:Najmudin;given-names:Shabir</infon><infon key="name_11">surname:Baslé;given-names:Arnaud</infon><infon key="name_12">surname:Cuskin;given-names:Fiona</infon><infon key="name_2">surname:Brás;given-names:Joana L. A.</infon><infon key="name_3">surname:Jackson;given-names:Adam</infon><infon key="name_4">surname:Rogowski;given-names:Artur</infon><infon key="name_5">surname:Gray;given-names:Joseph</infon><infon key="name_6">surname:Yadav;given-names:Madhav P.</infon><infon key="name_7">surname:Henrissat;given-names:Bernard</infon><infon key="name_8">surname:Fontes;given-names:Carlos M. G. A.</infon><infon key="name_9">surname:Gilbert;given-names:Harry J.</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">291</infon><infon key="year">2016</infon><offset>0</offset><text>The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans*</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>79</offset><text>The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1757</offset><text>Introduction</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1770</offset><text>The plant cell wall is an important biological substrate. This complex composite structure is depolymerized by microorganisms that occupy important highly competitive ecological niches, whereas the process makes an important contribution to the carbon cycle. Lignocellulosic degradation is also of continued interest to environmentally sensitive industries such as the biofuels and biorefinery sectors, where the use of sustainable or renewable substrates is of increasing importance. Given that the plant cell wall is the most abundant source of renewable organic carbon on the planet, this macromolecular substrate has substantial industrial potential.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2425</offset><text>An example of the chemical complexity of the plant cell wall is provided by xylan, which is the major hemicellulosic component. This polysaccharide comprises a backbone of β-1,4-d-xylose residues in their pyranose configuration (Xylp) that are decorated at O2 with 4-O-methyl-d-glucuronic acid (GlcA) and at O2 and/or O3 with α-l-arabinofuranose (Araf) residues, whereas the polysaccharide can also be extensively acetylated. In addition, the Araf side chain decorations can also be esterified to ferulic acid that, in some species, provide a chemical link between hemicellulose and lignin. The precise structure of xylans varies between plant species, in particular in different tissues and during cellular differentiation. In specialized plant tissues, such as the outer layer of cereal grains, xylans are extremely complex, and side chains may comprise a range of other sugars including l- and d-galactose and β- and α-Xylp units. Indeed, in these cereal brans, xylans have very few backbone Xylp units that are undecorated, and the side chains can contain up to six sugars.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3517</offset><text>Reflecting the chemical and physical complexity of the plant cell wall, microorganisms that utilize these composite structures express a large number of polysaccharide-degrading enzymes, primarily glycoside hydrolases, but also polysaccharide lyases, carbohydrate esterases, and lytic polysaccharide monooxygenases. These carbohydrate active enzymes are grouped into sequence-based families in the CAZy database. With respect to xylan degradation, the backbone of simple xylans is hydrolyzed by endo-acting xylanases, the majority of which are located in glycoside hydrolase (GH)5 families GH10 and GH11, although they are also present in GH8. The extensive decoration of the xylan backbone generally restricts the capacity of these enzymes to attack the polysaccharide prior to removal of the side chains by a range of α-glucuronidases, α-arabinofuranosidases, and esterases. Two xylanases, however, utilize the side chains as essential specificity determinants and thus target decorated forms of the hemicellulose. The GH30 glucuronoxylanases require the Xylp bound at the −2 to contain a GlcA side chain (the scissile bond targeted by glycoside hydrolases is between subsites −1 and +1, and subsites that extend toward the non-reducing and reducing ends of the substrate are assigned increasing negative and positive numbers, respectively). The GH5 arabinoxylanase (CtXyl5A) derived from Clostridium thermocellum displays an absolute requirement for xylans that contain Araf side chains. In this enzyme, the key specificity determinant is the Araf appended to O3 of the Xylp bound in the active site (−1 subsite). The reaction products generated from arabinoxylans, however, suggest that Araf can be accommodated at subsites distal to the active site.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5280</offset><text>CtXyl5A is a multimodular enzyme containing, in addition to the GH5 catalytic module (CtGH5); three non-catalytic carbohydrate binding modules (CBMs) belonging to families 6 (CtCBM6), 13 (CtCBM13), and 62 (CtCBM62); fibronectin type 3 (Fn3) domain; and a C-terminal dockerin domain Fig. 1. Previous studies of Fn3 domains have indicated that they might function as ligand-binding modules, as a compact form of peptide linkers or spacers between other domains, as cellulose-disrupting modules, or as proteins that help large enzyme complexes remain soluble. The dockerin domain recruits the enzyme into the cellulosome, a multienzyme plant cell wall degrading complex presented on the surface of C. thermocellum. CtCBM6 stabilizes CtGH5, and CtCBM62 binds to d-galactopyranose and l-arabinopyranose. The function of the CtCBM13 and Fn3 modules remains unclear. Similarly, the mechanism of substrate recognition and its impact on specificity are key unresolved issues. This report exploits the crystal structure of mature CtXyl5A lacking its C-terminal dockerin domain (CtXyl5A-Doc), and the enzyme in complex with ligands, to explore the mechanism of substrate specificity. The data show that the plasticity in substrate recognition enables the enzyme to hydrolyze highly complex xylans that are not accessible to classical GH10 and GH11 endo-xylanases.</text></passage><passage><infon key="file">zbc0441653440001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>6633</offset><text>Molecular architecture of GH5_34 enzymes. Modules prefaced by GH, CBM, or CE are modules in the indicated glycoside hydrolase, carbohydrate binding module, or carbohydrate esterase families, respectively. Laminin_3_G domain belongs to the concanavalin A lectin superfamily, and FN3 denotes a fibronectin type 3 domain. Segments labeled D are dockerin domains.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>6993</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>7001</offset><text>Substrate Specificity of CtXyl5A</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>7034</offset><text>Previous studies showed that CtXyl5A is an arabinoxylan-specific xylanase that generates xylooligosaccharides with an arabinose linked O3 to the reducing end xylose. The enzyme is active against both wheat and rye arabinoxylans (abbreviated as WAX and RAX, respectively). It was proposed that arabinose decorations make productive interactions with a pocket (−2*) that is abutted onto the active site or −1 subsite. Arabinose side chains of the other backbone xylose units in the oligosaccharides generated by CtXyl5A were essentially random. These data suggest that O3, and possibly O2, on the xylose residues at subsites distal to the active site and −2* pocket are solvent-exposed, implying that the enzyme can access highly decorated xylans. To test this hypothesis, the activity of CtXyl5A against xylans from cereal brans was assessed. CtXyl5a was incubated with a range of xylans for 16 h at 60 °C, and the limit products were visualized by TLC. These xylans are highly decorated not only with Araf and GlcA units but also with l-Gal, d-Gal, and d-Xyl. Indeed, very few xylose units in the backbone of bran xylans lack side chains. The data presented in Table 1 showed that CtXyl5A was active against corn bran xylan (CX). In contrast typical endo-xylanases from GH10 and GH11 were unable to attack CX, reflecting the lack of undecorated xylose units in the backbone (the active site of these enzymes can only bind to non-substituted xylose residues). The limit products generated by CtXyl5A from CX consisted of an extensive range of oligosaccharides. These data support the view that in subsites out with the active site the O2 and O3 groups of the bound xylose units are solvent-exposed and will thus tolerate decoration.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>8773</offset><text>Kinetics of GH5_34 arabinoxylanases</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>8809</offset><text>ND, not determined; NA, no activity.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th align="center" rowspan="2" colspan="1">Enzyme</th><th align="center" rowspan="2" colspan="1">Variant</th><th align="center" rowspan="1" colspan="3"><italic>k</italic><sub>cat</sub>/<italic>K<sub>m</sub></italic><hr/></th></tr><tr><th align="center" rowspan="1" colspan="1">WAX</th><th align="center" rowspan="1" colspan="1">RAX</th><th align="center" rowspan="1" colspan="1">CX</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="3"><italic>min</italic><sup>−<italic>1</italic></sup>
<italic>mg</italic><sup>−<italic>1</italic></sup>
<italic>ml</italic></td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3-CBM62</td><td align="left" rowspan="1" colspan="1">800</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">460</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13-Fn3</td><td align="left" rowspan="1" colspan="1">1,232</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">659</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6-CBM13</td><td align="left" rowspan="1" colspan="1">1,307</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">620</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6</td><td align="left" rowspan="1" colspan="1">488</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">102</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: E68A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: Y92A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N135A</td><td align="left" rowspan="1" colspan="1">260</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>Xyl5A</td><td align="left" rowspan="1" colspan="1"><italic>Ct</italic>GH5-CBM6: N139A</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td><td align="left" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Ac</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">628</td><td align="left" rowspan="1" colspan="1">1,641</td><td align="left" rowspan="1" colspan="1">289</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Gp</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">2,600</td><td align="left" rowspan="1" colspan="1">9,986</td><td align="left" rowspan="1" colspan="1">314</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">Wild type</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td><td align="left" rowspan="1" colspan="1">ND</td></tr><tr><td align="left" rowspan="1" colspan="1"><italic>Vb</italic>GH5</td><td align="left" rowspan="1" colspan="1">D45A</td><td align="left" rowspan="1" colspan="1">102</td><td align="left" rowspan="1" colspan="1">203</td><td align="left" rowspan="1" colspan="1">23</td></tr></tbody></table>
</infon><offset>8846</offset><text>Enzyme Variant kcat/Km WAX RAX CX min−1mg−1ml CtXyl5A CtGH5-CBM6-CBM13-Fn3-CBM62 800 ND 460 CtXyl5A CtGH5-CBM6-CBM13-Fn3 1,232 ND 659 CtXyl5A CtGH5-CBM6-CBM13 1,307 ND 620 CtXyl5A CtGH5-CBM6 488 ND 102 CtXyl5A CtGH5-CBM6: E68A NA NA NA CtXyl5A CtGH5-CBM6: Y92A NA NA NA CtXyl5A CtGH5-CBM6: N135A 260 ND ND CtXyl5A CtGH5-CBM6: N139A NA NA NA AcGH5 Wild type 628 1,641 289 GpGH5 Wild type 2,600 9,986 314 VbGH5 Wild type ND ND ND VbGH5 D45A 102 203 23 </text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9333</offset><text>To explore whether substrate bound only at −2* and −1 in the negative subsites was hydrolyzed by CtXyl5A, the limit products of CX digested by the arabinoxylanase were subjected to size exclusion chromatography using a Bio-Gel P-2, and the smallest oligosaccharides (largest elution volume) were chosen for further study. HPAEC analysis of the smallest oligosaccharide fraction (pool 4) contained two species with retention times of 14.0 min (oligosaccharide 1) and 20.8 min (oligosaccharide 2) (Fig. 2). Positive mode electrospray mass spectrometry showed that pool 4 contained exclusively molecular ions with a m/z = 305 [M + Na]+, which corresponds to a pentose-pentose disaccharide (molecular mass = 282 Da) as a sodium ion adduct, whereas a dimer of the disaccharide with a sodium adduct (m/z = 587 [2M+Na]+) was also evident. The monosaccharide composition of pool 4 determined by TFA hydrolysis contained xylose and arabinose in a 3:1 ratio. This suggests that the two oligosaccharides consist of two disaccharides: one consisting of two xylose residues and the other consisting of an arabinose linked to a xylose. Treatment of pool 4 with the nonspecific arabinofuranosidase, CjAbf51A, resulted in the loss of oligosaccharide 2 and the production of both xylose and arabinose, indicative of a disaccharide of xylose and arabinose. Incubation of pool 4 with a β-1,3-xylosidase (XynB) converted oligosaccharide 1 into xylose, demonstrating that this molecule is the disaccharide β-1,3-xylobiose. This view is supported by the inability of a β-1,4-specific xylosidase to hydrolyze oligosaccharide 1 or oligosaccharide 2 (data not shown). The crucial importance of occupancy of the −2* pocket for catalytic competence is illustrated by the inability of the enzyme to hydrolyze linear β-1,4-xylooligosaccharides. The generation of Araf-Xylp and Xyl-β-1,3-Xyl as reaction products demonstrates that occupancy of the −2 subsite is not essential for catalytic activity, which is in contrast to all endo-acting xylanases where this subsite plays a critical role in enzyme activity. Indeed, the data demonstrate that −2* plays a more important role in productive substrate binding than the −2 subsite. Unfortunately, the inability to generate highly purified (Xyl-β-1,4)n-[β-1,3-Xyl/Ara]-Xyl oligosaccharides from arabinoxylans prevented the precise binding energies at the negative subsites to be determined.</text></passage><passage><infon key="file">zbc0441653440002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11762</offset><text>Identification of the disaccharide reaction products generated from CX. The smallest reaction products were purified by size exclusion chromatography and analyzed by HPAEC (A) and positive mode ESI-MS (B), respectively. The samples were treated with a nonspecific arabinofuranosidase (CjAbf51A) and a GH3 xylosidase (XynB) that targeted β-1,3-xylosidic bonds. X, xylose; A, arabinose. The m/z = 305 species denotes a pentose disaccharide as a sodium adduct [M + Na]+, whereas the m/z = 587 signal corresponds to an ESI-MS dimer of the pentose disaccharide also as a sodium adduct [2M + Na]+.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>12357</offset><text>Crystal Structure of the Catalytic Module of CtXyl5A in Complex with Ligands</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12434</offset><text>To understand the structural basis for the biochemical properties of CtXyl5A, the crystal structure of the enzyme with ligands that occupy the substrate binding cleft and the critical −2* subsite were sought. The data presented in Fig. 3A show the structure of the CtXyl5A derivative CtGH5-CtCBM6 in complex with arabinose bound in the −2* pocket. Interestingly, the bound arabinose was in the pyranose conformation rather than in its furanose form found in arabinoxylans. O1 was facing toward the active site −1 subsite, indicative of the bound arabinose being in the right orientation to be linked to the xylan backbone via an α-1,3 linkage. As discussed on below, the axial O4 of the Arap did not interact with the −2* subsite, suggesting that the pocket might be capable of binding a xylose molecule. Indeed, soaking apo crystals with xylose showed that the pentose sugar also bound in the −2* subsite in its pyranose conformation (Fig. 3B). These crystal structures support the biochemical data presented above showing that the enzyme generated β-1,3-xylobiose from CX, which would require the disaccharide to bind at the −1 and −2* subsites. A third product complex was generated by co-crystallizing the nucleophile inactive mutant CtGH5E279S-CtCBM6 with a WAX-derived oligosaccharide (Fig. 3C). The data revealed a pentasaccharide bound to the enzyme, comprising β-1,4-xylotetraose with an Araf linked α-1,3 to the reducing end xylose. The xylotetraose was positioned in subsites −1 to −4 and the Araf in the −2* pocket. Analysis of the three structures showed that O1, O2, O3, and the endocyclic oxygen occupied identical positions in the Arap, Araf, and Xylp ligands bound in the −2* subsite and thus made identical interactions with the pocket. O1 makes a polar contact with Nδ2 of Asn139, O2 is within hydrogen bonding distance with Oδ1 of Asn139 and the backbone N of Asn135, and O3 interacts with the N of Gly136 and Oϵ2 of Glu68. Although O4 of Arap does not make a direct interaction with the enzyme, O4 and O5 of Xylp and Araf, respectively, form hydrogen bonds with Oϵ1 of Glu68. Finally Tyr92 makes apolar parallel interactions with the pyranose or furanose rings of the three sugars.</text></passage><passage><infon key="file">zbc0441653440003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14671</offset><text>Representation of the residues involved in the ligands recognition at the −2* subsite. The protein backbone is represented as a cartoon in gray. Interacting residues are represented as stick in blue, and the catalytic residues and the mutated glutamate (into a serine) are in magenta. A, CtGH5-CBM6 in complex with an arabinopyranose. B, CtGH5-CBM6 in complex with a xylopyranose. C, CtGH5E279S-CBM6 in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). The xylan backbone is shown transparently for more clarity. Densities shown in blue are RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. The figure and all other structural figures were made with PyMOL unless otherwise stated.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15430</offset><text>The importance of the interactions between the ligands and the side chains of the residues in the −2* pocket were evaluated by alanine substitution of these amino acids. The mutants E68A, Y92A, and N139A were all inactive (Table 1), demonstrating the importance of the interactions of these residues with the substrate and reinforcing the critical role the −2* subsite plays in the activity of the enzyme. N135A retained wild type activity because the O2 of the sugars interacts with the backbone N of Asn135 and not with the side chain. Because the hydroxyls of Xylp or Araf in the −2* pocket are not solvent-exposed, the active site of the arabinoxylanase can only bind to xylose residues that contain a single xylose or arabinose O3 decoration. This may explain why the kcat/Km for CtXyl5A against WAX was 2-fold higher than against CX (Table 1). WAX is likely to have a higher concentration of single Araf decorations compared with CX and thus contain more substrate available to the arabinoxylanase.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16441</offset><text>In the active site of CtXyl5A the α-d-Xylp, which is in its relaxed 4C1 conformation, makes the following interactions with the enzyme (Fig. 4, A–C): O1 hydrogen bonds with the Nδ1 of His253 and Oϵ2 of Glu171 (catalytic acid-base) and makes a possible weak polar contact with the OH of Tyr255 and Oγ of Ser279 (mutation of the catalytic nucleophile); O2 hydrogen bonds with Nδ2 of Asn170 and OH of Tyr92. O3 (O1 of the Araf at the −2* subsite) makes a polar contact with Nδ2 of Asn139; the endocyclic oxygen hydrogens bonds with the OH of Tyr255. The Xylp in the active site makes strong parallel apolar interactions with Phe310. Substrate recognition in the active site is conserved between CtXyl5A and the closest GH5 structural homolog, the endoglucanase BaCel5A (PDB code 1qi2) as noted previously.</text></passage><passage><infon key="file">zbc0441653440004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>17270</offset><text>Comparison of the ligand recognition at the distal negative subsites between CtGH5E279S-CBM6, the cellulase BaCel5A, and the xylanase GH10.
A–C show CtGH5E279S-CBM6 is in complex with a pentasaccharide (β1,4-xylotetraose with an l-Araf linked α1,3 to the reducing end xylose). A, Poseview representation highlighting the hydrogen bonding and the hydrophobic interactions that occur in the negative subsites. C, density of the ligand shown in blue is RefMac maximum-likelihood σA-weighted 2Fo − Fc at 1.5 σ. D and E display BaCel5A in complex with deoxy-2-fluoro-β-d-cellotrioside (PDB code 1qi2), and F and G show CmXyn10B in complex with a xylotriose (PDB code 1uqy). The ligand are represented as sticks. B, D, and F are surface representations (CtGH5E279S-CBM6 in gray, BaCel5A in cyan, and the xylanase GH10 in light brown). C, E, and G show the protein backbone as a cartoon representation with the interacting residues represented as sticks. The black dashes represent the hydrogen bonds.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18283</offset><text>The capacity of CtXyl5A to act on the highly decorated xylan CX indicates that O3 and possibly O2 of the backbone Xylp units are solvent-exposed. This is consistent with the interaction of the xylotetraose backbone with the enzyme distal to the active site. A surface representation of the enzyme (Fig. 4B) shows that O3 and O2 of xylose units at subsites −2 to −4 are solvent-exposed and are thus available for decoration. Indeed, these pyranose sugars make very weak apolar interactions with the arabinoxylanase. At −2, Xylp makes planar apolar interactions with the Araf bound to the −2* subsite (Fig. 4C). Xylp at subsites −2 and −3, respectively, make weak hydrophobic contact with Val318, the −3 Xylp makes planar apolar interactions with Ala137, whereas the xylose at −4 forms parallel apolar contacts with Trp69. Comparison of the distal negative subsites of CtXyl5A with BaCel5A and a typical GH10 xylanase (CmXyn10B, PDB code 1uqy) highlights the paucity of interactions between the arabinoxylanase and its substrate out with the active site (Fig. 4). Thus, the cellulase contains three negative subsites and the sugars bound in the −2 and −3 subsites make a total of 9 polar interactions with the enzyme (Fig. 4, D and E). The GH10 xylanase also contains a −2 subsite that, similar to the cellulase, makes numerous interactions with the substrate (Fig. 4, F and G).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>19682</offset><text>The Influence of the Modular Architecture of CtXyl5A on Catalytic Activity</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>19757</offset><text>CtXyl5A, in addition to its catalytic module, contains three CBMs (CtCBM6, CtCBM13, and CtCBM62) and a fibronectin domain (CtFn3). A previous study showed that although the CBM6 bound in an exo-mode to xylo- and cellulooligosaccharides, the primary role of this module was to stabilize the structure of the GH5 catalytic module. To explore the contribution of the other non-catalytic modules to CtXyl5A function, the activity of a series of truncated derivatives of the arabinoxylanase were assessed. The data in Table 1 show that removal of CtCBM62 caused a modest increase in activity against both WAX and CX, whereas deletion of the Fn3 domain had no further impact on catalytic performance. Truncation of CtCBM13, however, caused a 4–5-fold reduction in activity against both substrates. Members of CBM13 have been shown to bind to xylans, mannose, and galactose residues in complex glycans, hinting that the function of CtCBM13 is to increase the proximity of substrate to the catalytic module of CtXyl5A. Binding studies, however, showed that CtCBM13 displayed no affinity for a range of relevant glycans including WAX, CX, xylose, mannose, galactose, and birchwood xylan (BX) (data not shown). It would appear, therefore, that CtCBM13 makes a structural contribution to the function of CtXyl5A.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>21061</offset><text>Crystal Structure of CtXyl5A-D</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21092</offset><text>To explore further the role of the non-catalytic modules in CtXyl5A the crystal structure of CtXyl5A extending from CtGH5 to CtCBM62 was sought. To obtain a construct that could potentially be crystallized, the protein was generated without the C-terminal dockerin domain because it is known to be unstable and prone to cleavage. Using this construct (CtXyl5A-D) the crystal structure of the arabinoxylanase was determined by molecular replacement to a resolution of 2.64 Å with Rwork and Rfree at 23.7% and 27.8%, respectively. The structure comprises a continuous polypeptide extending from Ala36 to Trp742 displaying four modules GH5-CBM6-CBM13-Fn3. Although there was some electron density for CtCBM62, it was not sufficient to confidently build the module (Fig. 5). Further investigation of the crystal packing revealed a large solvent channel adjacent to the area the CBM62 occupies. We postulate that the reason for the poor electron density is due to the CtCBM62 being mobile compared with the rest of the protein. The structures of CtGH5 and CtCBM6 have been described previously.</text></passage><passage><infon key="file">zbc0441653440005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>22184</offset><text>Surface representation of the tetra-modular arabinoxylanase and zoom view on the CtGH5 loop. The blue module is the CtGH5 catalytic domain, the green module corresponds to the CtCBM6, the yellow module is the CtCBM13, and the salmon module is the fibronectin domain. Surfaces are semitransparent with the protein backbone represented as a cartoon. The CtGH5 loop is stabilized between the CtCBM6 and the CtCBM13 modules. The black dashes represent the hydrogen bonds. The protein backbone is represented as cartoon, and interacting residues are shown as sticks.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22746</offset><text>CtCBM13 extends from Gly567 to Pro648. Typical of CBM13 proteins CtCBM13 displays a β-trefoil fold comprising the canonical pseudo 3-fold symmetry with a 3-fold repeating unit of 40–50 amino acid residues characteristic of the Ricin superfamily. Each repeat contains two pairs of antiparallel β-strands. A Dali search revealed structural homologs from the CBM13 family with an root mean square deviation less than 2.0 Å and sequence identities of less than 20% that include the functionally relevant homologs C. thermocellum exo-β-1,3-galactanase (PDB code 3vsz), Streptomyces avermitilis β-l-arabinopyranosidase (PDB code 3a21), Streptomyces lividans xylanase 10A (PDB code, 1mc9), and Streptomyces olivaceoviridis E-86 xylanase 10A (PDB code 1v6v).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>23509</offset><text>The Fn3 module displays a typical β-sandwich fold with the two sheets comprising, primarily, three antiparallel strands in the order β1-β2-β5 in β-sheet 1 and β4-β3-β6 in β-sheet 2. Although β-sheet 2 presents a cleft-like topology, typical of endo-binding CBMs, the surface lacks aromatic residues that play a key role in ligand recognition, and in the context of the full-length enzyme, the cleft abuts into CtCBM13 and thus would not be able to accommodate an extended polysaccharide chain (see below).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24045</offset><text>In the structure of CtXyl5A-D, the four modules form a three-leaf clover-like structure (Fig. 5). Between the interfaces of CtGH5-CBM6-CBM13 there are a number of interactions that maintain the modules in a fixed position relative to each other. The interaction of CtGH5 and CtCBM6, which buries a substantial apolar solvent-exposed surface of the two modules, has been described previously. The polar interactions between these two modules comprise 14 hydrogen bonds and 5 salt bridges. The apolar and polar interactions between these two modules likely explaining why they do not fold independently compared with other glycoside hydrolases that contain CBMs. CtCBM13 acts as the central domain, which interacts with CtGH5, CtCBM6, and CtFn3 via 2, 5, and 4 hydrogen bonds, respectively, burying a surface area of ∼450, 350, and 500 Å2, respectively, to form a compact heterotetramer. With respect to the CtCBM6-CBM13 interface, the linker (SPISTGTIP) between the two modules, extending from Ser514 to Pro522, adopts a fixed conformation. Such sequences are normally extremely flexible; however, the two Ile residues make extensive apolar contacts within the linker and with the two CBMs, leading to conformational stabilization. The interactions between CtGH5 and the two CBMs, which are mediated by the tip of the loop between β-7 and α-7 (loop 7) of CtGH5, not only stabilize the trimodular clover-like structure but also make a contribution to catalytic function. Central to the interactions between the three modules is Trp285, which is intercalated between the two CBMs. The Nϵ of this aromatic residue makes hydrogen bonds with the backbone carbonyl of Val615 and Gly616 in CtCBM13, and the indole ring makes several apolar contacts with CtCBM6 (Pro440, Phe489, Gly491, and Ala492) (Fig. 5). Indeed, loop 7 is completely disordered in the truncated derivative of CtXyl5A comprising CtGH5 and CtCBM6, demonstrating that the interactions with CtCBM13 stabilize the conformation of this loop. Although the tip of loop 7 does not directly contribute to the topology of the active site, it is only ∼12 Å from the catalytic nucleophile Glu279. Thus, any perturbation of the loop (through the removal of CtCBM13) is likely to influence the electrostatic and apolar environment of the catalytic apparatus, which could explain the reduction in activity associated with the deletion of CtCBM13.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>26451</offset><text>Similar to the interactions between CtCBM6 and CtCBM13, there are extensive hydrophobic interactions between CtCBM13 and CtFn3, resulting in very little flexibility between these modules. As stated above, the absence of CtCBM62 in the structure suggests that the module can adopt multiple positions with respect to the rest of the protein. The CtCBM62, by binding to its ligands (d-Galp and l-Arap) in plant cell walls, may be able to recruit the enzyme onto its target substrate. Xylans are not generally thought to contain such sugars. d-Galp, however, has been detected in xylans in the outer layer of cereal grains and in eucalyptus trees, which are substrates used by CtXyl5A. Thus, CtCBM62 may direct the enzyme to particularly complex xylans containing d-Galp at the non-reducing termini of the side chains, consistent with the open substrate binding cleft of the arabinoxylanase that is optimized to bind highly decorated forms of the hemicellulose. In general CBMs have little influence on enzyme activity against soluble substrates but have a significant impact on glycans within plant cell walls. Thus, the role of CBM62 will likely only be evident against insoluble composite substrates.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_4</infon><offset>27651</offset><text>Exploring GH5 Subfamily 34</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27678</offset><text>CtXyl5A is a member of a seven-protein subfamily of GH5, GH5_34. Four of these proteins are distinct, whereas the other three members are essentially identical (derived from different strains of C. thermocellum). To investigate further the substrate specificity within this subfamily, recombinant forms of three members of GH5_34 that were distinct from CtXyl5A were generated. AcGH5 has a similar molecular architecture to CtXyl5A with the exception of an additional carbohydrate esterase family 6 module at the C terminus (Fig. 1). The GH5_34 from Verrucomicrobiae bacterium, VbGH5, contains the GH5-CBM6-CBM13 core structure, but the C-terminal Fn3-CBM62-dockerin modules, present in CtXyl5A, are replaced with a Laminin_3_G domain, which, by analogy to homologous domains in other proteins that have affinity for carbohydrates, may display a glycan binding function. The Verrucomicobiae enzyme also has an N-terminal GH43 subfamily 10 (GH43_10) catalytic module. The fungal GH5_34, GpGH5, unlike the two bacterial homologs, comprises a single GH5 catalytic module lacking all of the other accessory modules (Fig. 1). GpGh5 is particularly interesting as Gonapodya prolifera is the only fungus of the several hundred fungal genomes that encodes a GH5_34 enzyme. In fact there are four potential GH5_34 sequences in the G. prolifera genome, all of which show high sequence homology to Clostridium GH5_34 sequences. G. prolifera and Clostridium occupy similar environments, suggesting that the GpGH5_34 gene was acquired from a Clostridium species, which was followed by duplication of the gene in the fungal genome. The sequence identity of the GH5_34 catalytic modules with CtXyl5A ranged from 55 to 80% (supplemental Fig. S1). All the GH5_34 enzymes were active on the arabinoxylans RAX, WAX, and CX but displayed no activity on BX (Table 1 and Fig. 6) and are thus defined as arabinoxylanases. The limit products generated by CtXyl5A, AcGH5, and GpGH5 comprised a range of oligosaccharides with some high molecular weight material. The oligosaccharides with low degrees of polymerization were absent in the VbGH5 reaction products. However, the enzyme generated a large amount of arabinose, which was not produced by the other arabinoxylanases. Given that GH43_10 is predominantly an arabinofuranosidase subfamily of GH43, the arabinose generated by VbGH5 is likely mediated by the N-terminal catalytic module (see below). Kinetic analysis showed that AcGH5 displayed similar activity to CtXyl5A against both WAX and RAX and was 2-fold less active against CX. When initially measuring the activity of wild type VbGH5 against the different substrates, no clear data could be obtained, regardless of the concentration of enzyme used the reaction appeared to cease after a few minutes. We hypothesized that the N-terminal GH43_10 rapidly removed single arabinose decorations from the arabinoxylans depleting the substrate available to the arabinoxylanase, explaining why this activity was short lived. To test this hypothesis, the conserved catalytic base (Asp45) of the GH43_10 module of VbGH5 was substituted with alanine, which is predicted to inactivate this catalytic module. The D45A mutant did not produce arabinose consistent with the arabinofuranosidase activity displayed by the GH43_10 module in the wild type enzyme (Fig. 6). The kinetics of the GH5_34 arabinoxylanase catalytic module was now measurable, and activities were determined to be between ∼6- and 10-fold lower than that of CtXyl5A. Interestingly, the fungal arabinoxylanase displays the highest activities against WAX and RAX, ∼4- and 6-fold higher, respectively, than CtXyl5A; however, there is very little difference in the activity between the eukaryotic and prokaryotic enzymes against CX. Attempts to express individual modules of a variety of truncations of AcGH5 and VbGH5 were unsuccessful. This may indicate that the individual modules can only fold correctly when incorporated into the full-length enzyme, demonstrating the importance of intermodule interactions to maintain the structural integrity of these enzymes.</text></passage><passage><infon key="file">zbc0441653440006.jpg</infon><infon key="id">F6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>31787</offset><text>Products profile generated of GH5_34 enzymes. The enzymes at 1 μm were incubated with the four different xylans at 1% in 50 mm sodium phosphate buffer for 16 h at 37 °C (GpGH5, VbGH5, and AcGH5) or 60 °C. The limit products were separated by TLC. The xylooligosaccharide standards (X) are indicated by their degrees of polymerization.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>32125</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>32136</offset><text>A characteristic feature of enzymes that attack the plant cell wall is their complex molecular architecture. The CBMs in these enzymes generally play a role in substrate targeting and are appended to the catalytic modules through flexible linker sequences. CtXyl5A provides a rare visualization of the structure of multiple modules within a single enzyme. The central feature of these data is the structural role played by two of the CBMs, CtCBM6 and CtCBM13, in maintaining the active conformation of the catalytic module, CtGH5. The crystallographic data described here are supported by biochemical data showing either that these two modules do not bind to glycans (CtCBM13) or that the recognition of the non-reducing end of xylan or cellulose chains (CtCBM6) is unlikely to be biologically significant. It should be emphasized, however, that glycan binding and substrate targeting may only be evident in the full-length enzyme acting on highly complex structures such as the plant cell wall, as observed recently by a CBM46 module in the Bacillus xyloglucanase/mixed linked glucanase BhCel5B.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>33233</offset><text>CtXyl5A is a member of GH5 that contains 6644 members. These proteins have been subdivided into 51 subfamilies based on sequence similarity. CtXyl5A is a member of subfamily GH5_34. Here we have explored the substrate specificity of the other members of this subfamily. Despite differences in sequence identity all of the homologs were shown to be arabinoxylanases. Consistent with the conserved substrate specificity, all members of GH5_34 contained the specificity determinants Glu68, Tyr92, and Asn139, which make critical interactions with the xylose or arabinose in the −2* subsite, which are 1,3-linked to the xylose positioned in the active site. The presence of a CBM62 in CtXyl5A and AcGH5 suggests that these enzymes target highly complex xylans that contain d-galactose in their side chains. The absence of a “non-structural” CBM in GpGH5 may indicate that this arabinoxylanase is designed to target simpler arabinoxylans present in the endosperm of cereals. Although the characterization of all members of GH5_34 suggests that this subfamily is monospecific, differences in specificity are observed in other subfamilies of GHs including GH43 and GH5. Thus, as new members of GH5_34 are identified from genomic sequence data and subsequently characterized, the specificity of this family may require reinterpretation.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>34568</offset><text>An intriguing feature of VbGH5 is that the limited products generated by this enzymes are much larger than those produced by the other arabinoxylanases. This suggests that although arabinose decorations contribute to enzyme specificity (VbGH5 is not active on xylans lacking arabinose side chains), the enzyme requires other specificity determinants that occur less frequently in arabinoxylans. This has some resonance with a recently described GH98 xylanase that also exploits specificity determinants that occur infrequently and are only evident in highly complex xylans (e.g. CX).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>35152</offset><text>To conclude, this study provides the molecular basis for the specificity displayed by arabinoxylanases. Substrate specificity is dominated by the pocket that binds single arabinose or xylose side chains. The open xylan binding cleft explains why the enzyme is able to attack highly decorated forms of the hemicellulose. It is also evident that appending additional catalytic modules and CBMs onto the core components of these enzymes generates bespoke arabinoxylanases with activities optimized for specific functions. The specificities of the arabinoxylanases described here are distinct from the classical endo-xylanases and thus have the potential to contribute to the toolbox of biocatalysts required by industries that exploit the plant cell wall as a sustainable substrate.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>35932</offset><text>Experimental Procedures</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>35956</offset><text>Cloning, Expression, and Purification of Components of CtXyl5A</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36019</offset><text>All recombinant forms of CtXyl5A used in this study were expressed in the cytoplasm of Escherichia coli because they lacked a signal peptide. DNA encoding CtGH5-CtCBM6 and CtXyl5A-D (CtXyl5A lacking the C-terminal dockerin domain (CtGH5-CtCBM6-CtCBM13-Fn3-CtCBM62)) were described previously. DNA encoding CtGH5-CtCBM6-CtCBM13-Fn3 and CtGH5-CtCBM6-CtCBM13 and mature Acetivibrio cellulolyticus GH5 (AcGH5) were amplified by PCR using plasmid encoding the full-length C. thermocellum arabinoxylanase or A. cellulolyticus genomic DNA as the respective templates. DNA encoding the G. prolifera GH5 (GpGH5) and V. bacterium GH5 (VbGH5) were initially generated by GeneArt® gene synthesis (Thermo Fisher Scientific). DNA encoding VbGH5 lacking the C-terminal cell surface anchoring residues was also amplified by PCR using the synthesized nucleic acid as the template. All the primers used in the PCRs required restriction sites and plasmids used are listed inj supplemental Table S1. All constructs were cloned such that the encoded proteins contain a C-terminal His6 tag. Site-directed mutagenesis was carried out using the PCR-based QuikChange method (Stratagene) deploying the primers listed in supplemental Table S1.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37237</offset><text>To express the recombinant proteins, E. coli strain BL21(DE3), harboring appropriate recombinant plasmids, was cultured to mid-exponential phase in Luria broth at 37 °C. Isopropyl β-d-galactopyranoside at 1 mm was then added to induce recombinant gene expression, and the culture incubated for a further 18 h at 16 °C. The recombinant proteins were purified to >90% electrophoretic purity by immobilized metal ion affinity chromatography using TalonTM (Clontech), cobalt-based matrix, and elution with 100 mm imidazole, as described previously. When preparing the selenomethionine derivative of CtXyl5A-D for crystallography, the proteins were expressed in E. coli B834 (DE3), a methionine auxotroph, cultured in medium comprising 1 liter of SelenoMet Medium BaseTM, 50 ml of SelenoMetTM nutrient mix (Molecular Dimensions), and 4 ml of a 10 mg/ml solution of l-selenomethionine. Recombinant gene expression and protein purification were as described above except that all purification buffers were supplemented with 10 mm β-mercaptoethanol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>38285</offset><text>Enzyme Assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38299</offset><text>CtXyl5A-D and its derivatives were assayed for enzyme activity using the method of Miller to detect the release of reducing sugar. The standard assay was carried out in 50 mm sodium phosphate buffer, pH 7.0, containing 0.1 mg/ml BSA and at substrate concentrations ranging from 1 to 6 mg/ml. The pH and temperature optima were previously determined to be 7 and 60 °C, respectively, for the CtXyl5A-D and its derivatives. The optimum temperature for the other enzymes was found to be 37 °C, and pH optima of 5, 7, and 4 were determined for AcGH5, GpGH5 and VbGH5, respectively. All enzymes were assayed for activity at their individual temperature and pH optimum. A FLUOstar Omega microplate reader (BMG Labtech) was used to measure activity in 96-well plates. Overnight assays to assess end point products were carried out with 6 mg/ml substrate and 1 μm enzyme concentrations. The identification of potential reaction products was also assessed by HPAEC or TLC using methodology described previously.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>39304</offset><text>Oligosaccharide Analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39329</offset><text>Approximately 5 g of CX or WAX were digested to completion (no further increase in reducing sugar and change in the HPAEC product profile) with 3 μm of CtXyl5A-D at 60 °C for 48 h. The oligosaccharide products were purified by size exclusion chromatography using a Bio-Gel P2 column as described previously. The structures of the oligosaccharides were analyzed by positive ion-mode infusion/offline electrospray ionization (ESI)-MS following either dilution with 30% acetonitrile or via desalting as described previously </text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_4</infon><offset>39853</offset><text>Crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39869</offset><text>Purified SeMet CtXyl5A-D was concentrated and stored in 5 mm DTT, 2 mm CaCl2. Crystals of seleno-l-methionine-containing protein were obtained by hanging drop vapor diffusion in 40% (v/v) 2-methyl-2,4-pentandiol. The data were collected on Beamlines ID14-1 and ID14-4 at the European Synchrotron Radiation Facility (Grenoble, France) to a resolution of 2.64 Å. The data were processed using the programs iMOSFLM and SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994). The crystal belongs to the orthorhombic space group (P21212). The structure was solved by molecular replacement using independently solved structures of some of the modules of the CtXyl5A: CtGH5-CBM6 (PDB code 2y8k), Fn3 (PDB code 3mpc), and CtCBM62 (PDB codes 2y8m, 2yfz, and 2y9s) using PHASER. The CtCBM13 domain was built de novo. BUCCANEER and PHENIX were initially used for auto building. The structure was completed by iterative cycles of manual rebuilding in COOT in tandem with refinement with RefMac5. The final values for Rwork and Rfree) were 23.73 and 27.80%) using TLS and restraining refinement to amino acid residues 36–373 representing the CtGH5 module, 374–516 for the CtCBM6, 517–652 for CtCBM13, and 653–742 for CtFn3. Stereochemistry was assessed with COOT and PDBSUM (with 677 residues (96%) in preferred, 22 in allowed regions (3%), and 6 outliers (1%) in the Ramachandran plot).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41279</offset><text>To obtain structures of CtGH5-CBM6 in complex with ligand the protein was crystallized using the sitting drop vapor phase diffusion method with an equal volume (100 nl) of protein and reservoir solution (unless otherwise stated), using the robotic nanodrop dispensing systems (mosquitoR LCP; TTPLabTech). Crystals of the protein (10 mg/ml) co-crystallized with arabinose (300 mm) were obtained in 1 m ammonium sulfate, 0.1 m Bis-Tris, pH 5.5, and 1% PEG 3350. Crystals with xylose (300 mm) grew in 100 mm sodium/potassium phosphate, 100 mm MES, pH 6.5, and 2 m sodium chloride. To obtain crystals of the arabinoxylanase in complex with an oligosaccharide, the nucleophile mutant E279S was used and mixed with a range of arabinoxylooligosaccharides that was generated by digestion of WAX with CtGH5-CBM6 (see above) and thereafter by 100 nm of the Cellvibrio japonicus GH43 exo-1,4-β-xylosidase. Only the inclusion of the largest purified oligosaccharide generated crystals of the arabinoxylanase. Crystals of CtGH5E279S-CBM6 were obtained by mixing an equal volume (100 nl) of the protein (11 mg/ml)/oligosaccharide (10 mm) solution and mother liquor solution consisting of 100 mm Tris-Bicine, pH 8.5, 12.5% (w/v) polyethylene glycol with an average molecular mass of 1,000 Da, 12.5% (w/v) polyethylene glycol with an average molecular mass of 3,350 Da and 12.5% (R,S)-2-methyl-2,4-pentanediol (racemic). Crystallographic data were collected on Beamlines IO2, IO4-1, and I24 at the DIAMOND Light Source (Harwell, UK). The data were processed using XDS The crystal structures were solved by molecular replacement using MolRep with CtGH5-CtCBM6 (PDB code 5AK1) as the search model. The refinement was done in RefMac5, and COOT was used for model (re)building. The final model were validated using Molprobity. The data collection and refinement statistics are listed in Table 2.</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>43158</offset><text>Data collection and refinement statistics</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table_caption</infon><offset>43200</offset><text>The values in parentheses are for highest resolution shell.</text></passage><passage><infon key="file">T2.xml</infon><infon key="id">T2</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table xmlns:xlink="http://www.w3.org/1999/xlink" frame="hsides" rules="groups"><thead valign="bottom"><tr><th rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">CtXyl5A<sub>-D</sub></th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Arap</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6-<italic>Xylp</italic></th><th align="center" rowspan="1" colspan="1">GH5-CBM6- (<italic>Araf</italic>-Xyl<italic>p</italic><sub>4</sub>)</th></tr></thead><tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data collection</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Source</td><td align="left" rowspan="1" colspan="1">ESRF-ID14-1</td><td align="left" rowspan="1" colspan="1">Diamond I04–1</td><td align="left" rowspan="1" colspan="1">Diamond I24</td><td align="left" rowspan="1" colspan="1">Diamond I02</td></tr><tr><td align="left" rowspan="1" colspan="1"> Wavelength (Å)</td><td align="left" rowspan="1" colspan="1">0.9334</td><td align="left" rowspan="1" colspan="1">0.9173</td><td align="left" rowspan="1" colspan="1">0.9772</td><td align="left" rowspan="1" colspan="1">0.9791</td></tr><tr><td align="left" rowspan="1" colspan="1"> Space group</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2</td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td><td align="left" rowspan="1" colspan="1">P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" rowspan="1" colspan="1"> Cell dimensions</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="left" rowspan="1" colspan="1">147.4, 191.7, 50.7</td><td align="left" rowspan="1" colspan="1">67.1, 72.4, 109.1</td><td align="left" rowspan="1" colspan="1">67.9, 72.5, 109.5</td><td align="left" rowspan="1" colspan="1">76.3, 123.2, 125.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> α, β, γ (°)</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td><td align="left" rowspan="1" colspan="1">90, 90, 90</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. of measured reflections</td><td align="left" rowspan="1" colspan="1">244,475 (29,324)</td><td align="left" rowspan="1" colspan="1">224,842 (11,281)</td><td align="left" rowspan="1" colspan="1">152,004 (4,996)</td><td align="left" rowspan="1" colspan="1">463,237 (23,068)</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. of independent reflections</td><td align="left" rowspan="1" colspan="1">42246 (5,920)</td><td align="left" rowspan="1" colspan="1">63,523 (3,175)</td><td align="left" rowspan="1" colspan="1">42,716 (2,334)</td><td align="left" rowspan="1" colspan="1">140,288 (6,879)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Resolution (Å)</td><td align="left" rowspan="1" colspan="1">50.70–2.64 (2.78–2.64)</td><td align="left" rowspan="1" colspan="1">44.85–1.65 (1.68–1.65)</td><td align="left" rowspan="1" colspan="1">45.16–1.90 (1.94–1.90)</td><td align="left" rowspan="1" colspan="1">48.43–1.65 (1.68–1.65)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>merge</sub> (%)</td><td align="left" rowspan="1" colspan="1">16.5 (69.5)</td><td align="left" rowspan="1" colspan="1">6.7 (65.1)</td><td align="left" rowspan="1" colspan="1">2.8 (8.4)</td><td align="left" rowspan="1" colspan="1">5.7 (74.9)</td></tr><tr><td align="left" rowspan="1" colspan="1"> CC<sub>1/2</sub></td><td align="left" rowspan="1" colspan="1">0.985 (0.478)</td><td align="left" rowspan="1" colspan="1">0.998 (0.594)</td><td align="left" rowspan="1" colspan="1">0.999 (0.982)</td><td align="left" rowspan="1" colspan="1">0.998 (0.484)</td></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>I</italic>/σ<italic>I</italic></td><td align="left" rowspan="1" colspan="1">8.0 (2.0)</td><td align="left" rowspan="1" colspan="1">13 (1.6)</td><td align="left" rowspan="1" colspan="1">26.6 (8.0)</td><td align="left" rowspan="1" colspan="1">11.2 (1.6)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Completeness (%)</td><td align="left" rowspan="1" colspan="1">98.5 (96.4)</td><td align="left" rowspan="1" colspan="1">98.5 (99.4)</td><td align="left" rowspan="1" colspan="1">98.6 (85.0)</td><td align="left" rowspan="1" colspan="1">98.8 (99.4)</td></tr><tr><td align="left" rowspan="1" colspan="1"> Redundancy</td><td align="left" rowspan="1" colspan="1">5.8 (5.0)</td><td align="left" rowspan="1" colspan="1">3.5 (3.6)</td><td align="left" rowspan="1" colspan="1">3.6 (2.1)</td><td align="left" rowspan="1" colspan="1">3.3 (3.4)</td></tr><tr><td colspan="5" rowspan="1"><hr/></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold>Refinement</bold></td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="left" rowspan="1" colspan="1">23.7/27.8</td><td align="left" rowspan="1" colspan="1">12.2/17.0</td><td align="left" rowspan="1" colspan="1">12.9/16.1</td><td align="left" rowspan="1" colspan="1">14.5/19.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> No. atoms</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Protein</td><td align="left" rowspan="1" colspan="1">5446</td><td align="left" rowspan="1" colspan="1">3790</td><td align="left" rowspan="1" colspan="1">3729</td><td align="left" rowspan="1" colspan="1">7333</td></tr><tr><td align="left" rowspan="1" colspan="1"> Ligand</td><td align="left" rowspan="1" colspan="1">19</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">20</td><td align="left" rowspan="1" colspan="1">92</td></tr><tr><td align="left" rowspan="1" colspan="1"> Water</td><td align="left" rowspan="1" colspan="1">227</td><td align="left" rowspan="1" colspan="1">579</td><td align="left" rowspan="1" colspan="1">601</td><td align="left" rowspan="1" colspan="1">923</td></tr><tr><td align="left" rowspan="1" colspan="1"> B-factors</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Protein</td><td align="left" rowspan="1" colspan="1">41.6</td><td align="left" rowspan="1" colspan="1">17.8</td><td align="left" rowspan="1" colspan="1">15.8</td><td align="left" rowspan="1" colspan="1">21.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Ligand</td><td align="left" rowspan="1" colspan="1">65.0</td><td align="left" rowspan="1" colspan="1">19.4</td><td align="left" rowspan="1" colspan="1">24.2</td><td align="left" rowspan="1" colspan="1">39.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> Water</td><td align="left" rowspan="1" colspan="1">35.4</td><td align="left" rowspan="1" colspan="1">38.5</td><td align="left" rowspan="1" colspan="1">32.2</td><td align="left" rowspan="1" colspan="1">37.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> R.m.s deviations</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Bond lengths (Å)</td><td align="left" rowspan="1" colspan="1">0.008</td><td align="left" rowspan="1" colspan="1">0.015</td><td align="left" rowspan="1" colspan="1">0.012</td><td align="left" rowspan="1" colspan="1">0.012</td></tr><tr><td align="left" rowspan="1" colspan="1"> Bond angles (°)</td><td align="left" rowspan="1" colspan="1">1.233</td><td align="left" rowspan="1" colspan="1">1.502</td><td align="left" rowspan="1" colspan="1">1.624</td><td align="left" rowspan="1" colspan="1">1.554</td></tr><tr><td align="left" rowspan="1" colspan="1"> Protein Data Bank code</td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5G56">5G56</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA0">5LA0</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="5LA1">5LA1</ext-link></td><td align="left" rowspan="1" colspan="1"><ext-link ext-link-type="pdb" xlink:href="2LA2">2LA2</ext-link></td></tr></tbody></table>
</infon><offset>43260</offset><text> CtXyl5A-D GH5-CBM6-Arap GH5-CBM6-Xylp GH5-CBM6- (Araf-Xylp4) Data collection Source ESRF-ID14-1 Diamond I04–1 Diamond I24 Diamond I02 Wavelength (Å) 0.9334 0.9173 0.9772 0.9791 Space group P21212 P212121 P212121 P212121 Cell dimensions a, b, c (Å) 147.4, 191.7, 50.7 67.1, 72.4, 109.1 67.9, 72.5, 109.5 76.3, 123.2, 125.4 α, β, γ (°) 90, 90, 90 90, 90, 90 90, 90, 90 90, 90, 90 No. of measured reflections 244,475 (29,324) 224,842 (11,281) 152,004 (4,996) 463,237 (23,068) No. of independent reflections 42246 (5,920) 63,523 (3,175) 42,716 (2,334) 140,288 (6,879) Resolution (Å) 50.70–2.64 (2.78–2.64) 44.85–1.65 (1.68–1.65) 45.16–1.90 (1.94–1.90) 48.43–1.65 (1.68–1.65) Rmerge (%) 16.5 (69.5) 6.7 (65.1) 2.8 (8.4) 5.7 (74.9) CC1/2 0.985 (0.478) 0.998 (0.594) 0.999 (0.982) 0.998 (0.484) I/σI 8.0 (2.0) 13 (1.6) 26.6 (8.0) 11.2 (1.6) Completeness (%) 98.5 (96.4) 98.5 (99.4) 98.6 (85.0) 98.8 (99.4) Redundancy 5.8 (5.0) 3.5 (3.6) 3.6 (2.1) 3.3 (3.4) Refinement Rwork/Rfree 23.7/27.8 12.2/17.0 12.9/16.1 14.5/19.9 No. atoms Protein 5446 3790 3729 7333 Ligand 19 20 20 92 Water 227 579 601 923 B-factors Protein 41.6 17.8 15.8 21.0 Ligand 65.0 19.4 24.2 39.5 Water 35.4 38.5 32.2 37.6 R.m.s deviations Bond lengths (Å) 0.008 0.015 0.012 0.012 Bond angles (°) 1.233 1.502 1.624 1.554 Protein Data Bank code 5G56 5LA0 5LA1 2LA2 </text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">title_1</infon><offset>45145</offset><text>Author Contributions</text></passage><passage><infon key="section_type">AUTH_CONT</infon><infon key="type">paragraph</infon><offset>45166</offset><text>A. L. obtained crystals of the GH5-CBM6 complex. L. I. C. analyzed the biochemistry of GH5 subfamilies. J. L. A. B. obtained crystals of CtXyl5A-D. A. J. analyzed the biochemistry of GH5-CBM6 and obtained crystals of GH5-CBM6. A. R. analyzed the biochemistry of GH5-CBM6 products. J. G. performed mass spectrometry. M. P. Y. provided the substrate. B. H. performed analysis of GH5 sequences. C. M. G. A. F. designed the experiments. H. J. G. designed the experiments, analyzed data, and contributed to writing the paper. S. N. solved the structure of CtXyl5A-D and contributed to writing the paper. A. B. used crystallography to solve GH5-CBM6 structures. F. C. analyzed the biochemistry of GH5-CBM6 mutants, obtained crystals of GH5-CBM6, and contributed to writing the paper.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>45944</offset><text>Supplementary Material</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>45967</offset><text>This work was supported in part by European Research Council Grant 322820 (to H. J. G. and B. H.), Biotechnology and Biological Research Council Grants BB/K020358/1 and BB/K001949/1 (to H. J. G.), Wellcome Trust Grant RES/0120/7613 (to H. J. G.), Agence Nationale de la Recherche Grant ANR 12-BIME-0006-01 (to B. H.), and Fundação para a Ciência e Tecnologia Grants PTDC/BIAPRO/103980/2008 and PTDC/BIAMIC/5947/2014 (to C. M. G. A. F.). The authors declare that they have no conflicts of interest with the contents of this article.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46502</offset><text>This article contains supplemental Table S1 and Fig. S1.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46559</offset><text>GH</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46562</offset><text>glycoside hydrolase</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46582</offset><text>CtXyl5A</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46590</offset><text>C. thermocellum arabinoxylanase</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46622</offset><text>CBM</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46626</offset><text>non-catalytic carbohydrate binding module</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46668</offset><text>Fn</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46671</offset><text>fibronectin</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46683</offset><text>WAX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46687</offset><text>wheat arabinoxylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46706</offset><text>RAX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46710</offset><text>rye arabinoxylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46727</offset><text>CX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46730</offset><text>corn bran xylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46746</offset><text>HPAEC</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46752</offset><text>high performance anion exchange chromatography</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46799</offset><text>PDB</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46803</offset><text>Protein Data Bank</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46821</offset><text>BX</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46824</offset><text>birchwood xylan</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46840</offset><text>ESI</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46844</offset><text>electrospray ionization.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>46869</offset><text>The abbreviations used are: </text></passage><passage><infon key="section_type">REF</infon><infon key="type">title</infon><offset>46898</offset><text>References</text></passage><passage><infon key="fpage">444</infon><infon key="lpage">455</infon><infon key="name_0">surname:Gilbert;given-names:H. 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<collection><source>PMC</source><date>20230815</date><key>pmc.key</key><document><id>5173035</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.18632/oncotarget.9692</infon><infon key="article-id_pmc">5173035</infon><infon key="article-id_pmid">27259995</infon><infon key="article-id_publisher-id">9692</infon><infon key="fpage">40965</infon><infon key="issue">27</infon><infon key="kwd">DNA N6-adenine methyltransferase M1.HpyAVI substrate recognition AdoMet-binding Helicobacter pylori Immunology and Microbiology Section Immune response Immunity</infon><infon key="license">This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</infon><infon key="lpage">40977</infon><infon key="name_0">surname:Ma;given-names:Bo</infon><infon key="name_1">surname:Ma;given-names:Ji</infon><infon key="name_2">surname:Liu;given-names:Dong</infon><infon key="name_3">surname:Guo;given-names:Ling</infon><infon key="name_4">surname:Chen;given-names:Huiling</infon><infon key="name_5">surname:Ding;given-names:Jingjin</infon><infon key="name_6">surname:Liu;given-names:Wei</infon><infon key="name_7">surname:Zhang;given-names:Hongquan</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Biochemical and structural characterization of a DNA N6-adenine methyltransferase from Helicobacter pylori </text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>108</offset><text>DNA N6-methyladenine modification plays an important role in regulating a variety of biological functions in bacteria. However, the mechanism of sequence-specific recognition in N6-methyladenine modification remains elusive. M1.HpyAVI, a DNA N6-adenine methyltransferase from Helicobacter pylori, shows more promiscuous substrate specificity than other enzymes. Here, we present the crystal structures of cofactor-free and AdoMet-bound structures of this enzyme, which were determined at resolutions of 3.0 Å and 3.1 Å, respectively. The core structure of M1.HpyAVI resembles the canonical AdoMet-dependent MTase fold, while the putative DNA binding regions considerably differ from those of the other MTases, which may account for the substrate promiscuity of this enzyme. Site-directed mutagenesis experiments identified residues D29 and E216 as crucial amino acids for cofactor binding and the methyl transfer activity of the enzyme, while P41, located in a highly flexible loop, playing a determinant role for substrate specificity. Taken together, our data revealed the structural basis underlying DNA N6-adenine methyltransferase substrate promiscuity.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">title_1</infon><offset>1269</offset><text>INTRODUCTION</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1282</offset><text>DNA methylation is a common form of modification on nucleic acids occurring in both prokaryotes and eukaryotes. Such a modification creates a signature motif recognized by DNA-interacting proteins and functions as a mechanism to regulate gene expression. DNA methylation is mediated by DNA methyltransferases (MTases), which catalyze the transfer of a methyl group from S-adenosyl-L- methionine (AdoMet) to a given position of a particular DNA base within a specific DNA sequence.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1763</offset><text>Three classes of DNA MTases have been identified to transfer a methyl group to different positions of DNA bases. C5-cytosine MTases, for example, methylate C5 of cytosine (m5C). In eukaryotes, m5C plays an important role in gene expression, chromatin organization, genome maintenance and parental imprinting, and is involved in a variety of human diseases including cancer. By contrast, the functions of the prokaryotic DNA cytosine MTase remain unknown. N4-cytosine MTases, which are frequently present in thermophilic or mesophilic bacteria, transfer a methyl group to the exocyclic amino group of cytosine (4mC). N4 methylation seems to be primarily a component of bacterial immune system against invasion by foreign DNA, such as conjugative plasmids and bacteriophages. The third group, N6-adenine MTases methylate the exocyclic amino groups of adenine (6mA), which exists in prokaryotes as a signal for genome defense, DNA replication and repair, regulation of gene expression, control of transposition and host-pathogen interactions. Recent studies utilizing new sequencing approaches have showed the existence of 6mA in several eukaryotic species. DNA 6mA modification is associated with important biological processes including nucleosome distribution close to the transcription start sites in Chlamydomonas, carrying heritable epigenetic information in C.elegans or controlling development of Drosophila.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3177</offset><text>All the three types of methylation exist in prokaryotes, and most DNA MTases are components of the restriction-modification (R-M) systems. The R-M systems are composed of two enzymes displaying opposing activities. “R” stands for a restriction endonuclease cleaving specific DNA sequences, while “M” symbolizes a modification methyltransferase rendering these sequences resistant to cleavage. The cooperation of these two enzymes provides a defensive mechanism to protect bacteria from infection by bacteriophages. The R-M systems are classified into three types based on specific structural features, position of DNA cleavage and cofactor requirements. In types I and III, the DNA adenine or cytosine methyltransferase is part of a multi-subunit enzyme that catalyzes both restriction and modification. By contrast, two separate enzymes exist in type II systems, where a restriction endonuclease and a DNA adenine or cytosine methyltransferase recognize the same targets.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4158</offset><text>To date, a number of bacterial DNA MTases have been structurally characterized, covering enzymes from all the three classes. All these MTases exhibit high similarity in their overall architectures, which are generally folded into two domains: a conserved larger catalytic domain comprising an active site for methyl transfer and a site for AdoMet-binding, and a smaller target (DNA)-recognition domain (TRD) containing variable regions implicated in sequence-specific DNA recognition and the infiltration of the DNA to flip the target base. Conserved amino acid motifs have been identified from reported structures, including ten motifs (I-X) in cytosine MTases and nine motifs (I-VIII and X) in adenine MTases, all of which are arranged in an almost constant order. According to the linear arrangement of three conserved domains, exocyclic amino MTases are subdivided into six groups (namely α, β, γ, ζ, δ and ε). N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Despite the considerable similarity among bacterial MTases, some differences were observed among the enzymes from various species. For example, the structural regions of MTases beyond the catalytic domain are rather variable, such as the C-terminal domain of M.TaqI, the extended arm of M.MboIIA and M.RsrI, the helix bundle of EcoDam, and so on.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>5553</offset><text>DNA methylation is thought to influence bacterial virulence. DNA adenine methyltransferase has been shown to play a crucial role in colonization of deep tissue sites in Salmonella typhimurium and Aeromonas hydrophila. Importantly, DNA adenine methylation is a global regulator of genes expressed during infection and inhibitors of DNA adenine methylation are likely to have a broad antimicrobial action. Dam was considered a promising target for antimicrobial drug development.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>6031</offset><text>Helicobacter pylori is a Gram-negative bacterium that persistently colonizes in human stomach worldwide. It is a major pathogen of gastritis and peptic ulcer diseases as well as a cancer-causing factor for gastric cancer. H. pylori is involved in 90% of all gastric malignancies, infecting nearly 50% of the world's population and is the most crucial etiologic agent for gastric adenocarcinoma. H. pylori strains possess a few R-M systems like other bacteria to function as defensive systems. H. pylori 26695, for example, has 23 R-M systems. Methyltransferases were suggested to be involved in H. pylori pathogenicity. M1.HpyAVI is a DNA adenine MTase that belongs to the type II R-M system. This system contains two DNA MTases named M1.HpyAVI and M2.HpyAVI, and a putative restriction enzyme. M1.HpyAVI encoded by ORF hp0050 is an N6-adenine methyltransferase belonging to the β-class MTase. It has been reported recently that this enzyme recognizes the sequence of 5′-GAGG-3′, 5′-GGAG-3′ or 5′-GAAG-3′ and methylates adenines in these sequences. Given that methylation of two adjacent adenines on the same strand have never been observed for other N6-adenine MTases, the methylation activity on 5′-GAAG-3′ seems to be a unique feature of M1.HpyAVI, compared with the homologs from other strains of H.pylori which is able to methylate only 5′-GAGG-3′. The structural basis and the catalytic mechanism underlying such a distinct activity are not well understood due to the lack of an available 3D structure of this enzyme.Here, we report the crystal structure of M1.HpyAVI from H. pylori 26695, which is the first determined N6-adenine MTase structure in H. pylori. The structure reveals a similar architecture as the canonical fold of homologous proteins, but displays several differences in the loop regions and TRD. Based on structural and biochemical analyses, we then identified two conserved amino acids, D29 at the catalytic site and E216 close to the C-terminus, as crucial residues for cofactor binding and methyltransferase activity of M1.HpyAVI. In addition, a non-conserved amino acid, P41, seems to play a key role in substrate recognition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>8207</offset><text>RESULTS</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8215</offset><text>Overall structure</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8233</offset><text>Recombinant full-length M1.HpyAVI was produced as a soluble protein in Escherichia coli, but was quite unstable and tended to aggregate in low salt environment. The protein, however, remained fully soluble in a buffer containing higher concentration of sodium chloride (>300 mM), which prompted that M1.HpyAVI is likely a halophilic protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8575</offset><text>The cofactor-free and AdoMet-bound proteins were crystallized at different conditions. Both structures were determined by means of molecular replacement, and refined to 3.0 Å and 3.1 Å, respectively. Statistics of X-ray data collection and structure refinement were summarized in Table 1.</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>8866</offset><text>Data collection and structure refinement statistics of M1.HpyAVI</text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="box" rules="all"><thead><tr><th align="left" valign="middle" rowspan="1" colspan="1"/><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI</th><th align="left" valign="middle" rowspan="1" colspan="1">M1.HpyAVI-AdoMet complex</th></tr></thead><tbody><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Data collection</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Wavelength (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.0000</td><td align="left" valign="middle" rowspan="1" colspan="1">0.97772</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Space group</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>4<sub>3</sub>2<sub>1</sub>2</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>P</italic>6<sub>5</sub></td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unit-cell parameters (Å, ˚)</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a</italic> = <italic>b</italic> = 69.73, <italic>c</italic> = 532.75<break/><italic>α = β = γ</italic> = 90</td><td align="left" valign="middle" rowspan="1" colspan="1"><italic>a = b</italic> = 135.60, <italic>c</italic> = 265.15<break/><italic>α = β</italic> = 90, <italic>γ</italic> = 120</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Resolution range (Å) <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">49.09-3.00 (3.09-3.00)</td><td align="left" valign="middle" rowspan="1" colspan="1">48.91-3.10 (3.18-3.10)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Unique reflections <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">27243</td><td align="left" valign="middle" rowspan="1" colspan="1">49833</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Multiplicity <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">3.7 (3.8)</td><td align="left" valign="middle" rowspan="1" colspan="1">5.6 (4.0)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Completeness (%)<xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">98.7 (98.9)</td><td align="left" valign="middle" rowspan="1" colspan="1">99.7 (97.8)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Mean <italic>I/δ</italic> (<italic>I</italic>) <xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">12.1 (3.4)</td><td align="left" valign="middle" rowspan="1" colspan="1">14.0 (1.9)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Solvent content (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">58.67</td><td align="left" valign="middle" rowspan="1" colspan="1">61.96</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>merge</sub>
<xref ref-type="table-fn" rid="tfn_001"><sup>a</sup></xref></td><td align="left" valign="middle" rowspan="1" colspan="1">0.073 (0.378)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.106 (0.769)</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Structure refinement</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>work</sub></td><td align="left" valign="middle" rowspan="1" colspan="1">0.251</td><td align="left" valign="middle" rowspan="1" colspan="1">0.221</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><italic>R</italic><sub>free</sub></td><td align="left" valign="middle" rowspan="1" colspan="1">0.308</td><td align="left" valign="middle" rowspan="1" colspan="1">0.276</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond lengths (Å)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td><td align="left" valign="middle" rowspan="1" colspan="1">0.007</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">R.m.s.d., bond angles (˚)</td><td align="left" valign="middle" rowspan="1" colspan="1">1.408</td><td align="left" valign="middle" rowspan="1" colspan="1">1.651</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1"><bold>Ramachandran plot</bold></td><td align="left" valign="middle" rowspan="1" colspan="1"/><td align="left" valign="middle" rowspan="1" colspan="1"/></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Favoured region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">89.44</td><td align="left" valign="middle" rowspan="1" colspan="1">91.44</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Allowed region (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">9.58</td><td align="left" valign="middle" rowspan="1" colspan="1">7.11</td></tr><tr><td align="left" valign="middle" rowspan="1" colspan="1">Outliers (%)</td><td align="left" valign="middle" rowspan="1" colspan="1">0.99</td><td align="left" valign="middle" rowspan="1" colspan="1">1.45</td></tr></tbody></table>
</infon><offset>8931</offset><text> M1.HpyAVI M1.HpyAVI-AdoMet complex Data collection Wavelength (Å) 1.0000 0.97772 Space group P43212 P65 Unit-cell parameters (Å, ˚) a = b = 69.73, c = 532.75α = β = γ = 90 a = b = 135.60, c = 265.15α = β = 90, γ = 120 Resolution range (Å) a 49.09-3.00 (3.09-3.00) 48.91-3.10 (3.18-3.10) Unique reflections a 27243 49833 Multiplicity a 3.7 (3.8) 5.6 (4.0) Completeness (%)a 98.7 (98.9) 99.7 (97.8) Mean I/δ (I) a 12.1 (3.4) 14.0 (1.9) Solvent content (%) 58.67 61.96 Rmergea 0.073 (0.378) 0.106 (0.769) Structure refinement Rwork 0.251 0.221 Rfree 0.308 0.276 R.m.s.d., bond lengths (Å) 0.007 0.007 R.m.s.d., bond angles (˚) 1.408 1.651 Ramachandran plot Favoured region (%) 89.44 91.44 Allowed region (%) 9.58 7.11 Outliers (%) 0.99 1.45 </text></passage><passage><infon key="file">T1.xml</infon><infon key="id">T1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>9745</offset><text>Values in parentheses are statistics of the highest resolution shell.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>9815</offset><text>Four and eight protein monomers resided in the asymmetric units of the two crystal structures. Some amino acids, particularly those within two loops (residues 32-61 and 152-172) in both structures, were poorly defined in electron density and had to be omitted from the refined models. Details of invisible amino acids are given in Table S1.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10156</offset><text>The two structures are very similar to each other (Figure 1) and could be well overlaid with an RMSD of 0.76 Å on 191 Cα atoms. The overall architecture of M1.HpyAVI revealed in these structures resembles the AdoMet-dependent MTase fold in which a twisted seven-stranded β-sheet flanked by six α-helices forms the structural core. Like the reported structures of the larger domain of MTases, three helices (αA, αB and αZ) are located at one face of the central β-sheet, while the other three αD, αE and αC sit at the other side. All these conserved structural motifs form a typical α/β Rossmann fold. The catalytic motif DPPY lies in a loop connecting αD and β4, and the cofactor AdoMet binds in a neighboring cavity. The loop (residues 136-166) located between β7 and αZ corresponds to a highly diverse region in other MTases that is involved in target DNA recognition. The hairpin loop (residues 101-133) bridging β6 and β7, which is proposed to bind DNA in the minor groove, displays a similar conformation as those observed in M.MboIIA, M.RsrI and M.pvuII. The missing loop (residues 33-58) in the structure of M1.HpyAVI corresponds to loop I in M.TaqI, which was also invisible in a structure without DNA. This loop, however, was well ordered in an M.TaqI-DNA complex structure and was shown to play a crucial role in DNA methylation by contacting the flipping adenine and recognizing specific DNA sequence.</text></passage><passage><infon key="file">oncotarget-07-40965-g001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>11634</offset><text>Overall structure of M1.HpyAVI</text></passage><passage><infon key="file">oncotarget-07-40965-g001.jpg</infon><infon key="id">F1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>11665</offset><text>A. Free form B. AdoMet-bound form. Ribbon diagram of M1.HpyAVI resembles an “AdoMet-dependent MTase fold”, a mixed seven-stranded β-sheet flanked by six α-helices, αA, αB, αZ on one side and αD, αE, αC on the other side, the cofactor AdoMet is bound in a cavity near the conserved enzyme activity motif DPPY. Rainbow coloring from blue through green to red indicates the N- to C-terminal position of the residues in the model. The α-helices and β-strands are labelled and numbered according to the commonly numbering rule for the known MTases. The AdoMet molecule is shown in green.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>12286</offset><text>Dimeric state of M1.HpyAVI in crystal and solution</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>12337</offset><text>Previous studies showed that some DNA MTases, e.g. M.BamHI and M.EcoRI, exist as monomer in solution, in agreement with the fact that a DNA substrate for a typical MTase is hemimethylated and therefore needs only a single methylation event to convert it into a fully methylated state. Increasing number of dimeric DNA MTases, however, has been identified from later studies. For instance, M.DpnII, M.RsrI, M.KpnI, and M.MboIIA have been found as dimers in solution. In addition, several MTases including M.MboIIA, M.RsrI and TTH0409 form tightly associated dimers in crystal structures. Nonetheless, some DNA MTases such as M.CcrMI and the Bacillus amyloliquefaciens MTase dissociate from dimer into monomer upon DNA-binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13063</offset><text>According to the arrangement of the three conserved domains, M1.HpyAVI belongs to the β-subgroup, in which a conserved motif NXXTX9−11AXRXFSXXHX4WX6−9 YXFXLX3RX9−26NPX1−6NVWX29−34A has been identified at the dimerization interface in crystal structures. Most of conserved amino acids within that motif are present in the sequence of M1.HpyAVI (Figure 2A), implying dimerization of this protein. In agreement, a dimer of M1.HpyAVI was observed in our crystal structures with the two monomers related by a two-fold axis (Figure 2B and 2C). An area of ~1900 Å2 was buried at the dimeric interface, taking up ca 17% of the total area. The dimeric architecture was greatly stabilized by hydrogen bonds and salt bridges formed among residues R86, D93 and E96. In addition, comparison of the dimer structure of M1.HpyAVI with some other β-class MTases (M1.MboIIA, M.RsrI and TTHA0409) suggested that the M1.HpyAVI dimer organized in a similar form as others (Figure S3).</text></passage><passage><infon key="file">oncotarget-07-40965-g002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>14040</offset><text>M1.HpyAVI exists as dimer in crystal and solution</text></passage><passage><infon key="file">oncotarget-07-40965-g002.jpg</infon><infon key="id">F2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>14090</offset><text>A. A conserved interface area of β-class MTases is defined in M1.HpyAVI. Residues that involved are signed in red color; Dimerization of free-form M1.HpyAVI B. and cofactor-bound M1.HpyAVI C. The two monomers are marked in green and blue, AdoMet molecules are marked in magenta. D. Gel-filtration analysis revealed that M1.HpyAVI exist as a dimer in solution. FPLC system coupled to a Superdex 75 10/300 column. Elution profiles at 280 nm (blue) and 260 nm (red) are: different concentration (0.05, 0.1, 0.2, 0.5 mg/ml) of M1.HpyAVI protein.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14635</offset><text>To probe the oligomeric form of M1.HpyAVI in solution, different concentrations of purified enzyme was loaded onto a Superdex 75 10/300 column. The protein was eluted at ~10 ml regardless of the protein concentrations, corresponding to a dimeric molecular mass of 54 kDa (Figure 2D).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>14919</offset><text>Our results clearly showed that M1.HpyAVI forms a dimer in both crystal and solution as other β-class MTases, which however disagrees with a previous investigation using dynamic light scattering (DLS) measurement and gel-filtration chromatography, suggesting that M1.HpyAVI is taking a monomeric state in solution. This variance might be caused by an addition of 100 mM arginine before cell lysis to keep protein solubility and also by later replacement of arginine with 30% glycerol by dialysis. These treatments probably changed protein conformation somehow and also the oligomeric state.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15513</offset><text>Structure comparisons</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15535</offset><text>As a β-class N6 adenine MTase, the M1.HpyAVI structure displayed a good similarity with M.MboIIA (PDB ID 1G60) and M.RsrI (PDB ID 1NW7), which are falling into the same subgroup. Superimposition of M1.HpyAVI onto them gave RMSDs of 1.63 Å and 1.9 Å on 168 and 190 Cα atoms, respectively. The most striking structural difference was found to locate on the TRD region (residues 133-163 in M1.HpyAVI) (Figure 3A–3C), where the secondary structures vary among these structures. By comparison with the other two enzymes that possess protruding arms containing several α-helices and/or β-strands, the TRD of M1.HpyAVI is much shorter in length (Figure S1), wrapping more closely around the structural core and lacking apparent secondary structures. Given the proposed role of the TRD for DNA interaction at the major groove, some differences of DNA recognition mode can be expected. Another difference locates at the highly flexible loop between β4 and αD (residues 33-58) of M1.HpyAVI, which was invisible in our structures but present in the structures of M.MboIIA and M.RsrI. Sequence alignment revealed that this region of M1.HpyAVI was longer than its counterparts by 13 and 16 amino acids respectively, which likely renders the H. pylori enzyme more flexible.</text></passage><passage><infon key="file">oncotarget-07-40965-g003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>16816</offset><text>Structural comparisons between M1.HpyAVI and other DNA MTases</text></passage><passage><infon key="file">oncotarget-07-40965-g003.jpg</infon><infon key="id">F3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>16878</offset><text>A. M1.HpyAVI; B. M.MboIIA; C. M.RsrI; D. TTHA0409; E. DpnM; F. M.TaqI. M1.HpyAVI possesses only a long disorder TRD region, compared with the structure-rich TRD of M.MboIIA, M.RsrI and TTHA0409, or the extra DNA-binding domain of DpnM and M.TaqI. The core structure is in cyan; TRD of M1.HpyAVI, M.MboIIA, M.RsrI and TTHA0409 is in red; The region between β4 and αD of M.MboIIA and M.RsrI is in green; DNA-binding domain of DpnM is in magenta; The C-terminal domain of M.TaqI is in orange.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17375</offset><text>N6-adenine and N4-cytosine MTases, in particular, are closely related by sharing common structural features. Structural comparison between M1.HpyAVI and a putative β-class N4 cytosine MTase named TTHA0409 (PDB ID 2ZIF) showed a good similarity as well, giving an RMSD of 1.73 Å on 164 Cα atoms (Figure 3D). Exactly like the above comparison, the most significant difference exists in the TRD, where the structures vary in terms of length and presence of α-helices (Figure S1).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>17863</offset><text>M1.HpyAVI displayed a considerable structural dissimilarity in comparison with N6-adenine MTases from other subgroups including the α-class DpnM (PDB ID 2DPM) and the γ-class M.TaqI (PDB ID 2ADM). Both comparisons gave RMSDs above 3.0 Å (Figure 3E and 3F). These two enzymes lack a counterpart loop present in the TRD of M1.HpyAVI, but instead rely on an extra domain for DNA binding and sequence recognition.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18281</offset><text>Collectively, M1.HpyAVI possesses a long disordered TRD, which is in sharp contrast to the secondary structure-rich TRD in other β-class N6 adenine or N4 cytosine MTases or the extra DNA binding domain present in DNA MTases from other subgroups. This striking difference may be a significant determinant of the wider substrate spectrum of this H. pylori enzyme.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>18646</offset><text>AdoMet-binding pocket</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18668</offset><text>The cofactor binding pocket of M1.HpyAVI is surrounded by residues 7-9, 29-31, 165-167, 216-218 and 221 (Figure 4A), which are conserved among most of DNA MTases. A hydrogen bond between D29 in the catalytic motif DPPY and the amino group of bound AdoMet is preserved as other MTase structures. Residues D8 and A9 from hydrogen-bonds with N6 and N1 of the purine ring, respectively, and E216 also locates at hydrogen bonding distance with O2′ and O3′ of the ribose. In addition, H168, T200 and S198 contact the terminal carboxyl of AdoMet. Superposition of M1.HpyAVI with the five structures shown in Figure 3 reveals that the orientation of cofactor is rather conserved except for M.TaqI (Figure 4B). The different conformation of the bound cofactor observed in M.TaqI might be attributable to the absence of corresponding residues of the conserved AdoMet-binding motif FXGXG in that structure.</text></passage><passage><infon key="file">oncotarget-07-40965-g004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>19568</offset><text>Structural and biochemical analyses define two conserved residues D29 and E216 to be the key sites for AdoMet binding</text></passage><passage><infon key="file">oncotarget-07-40965-g004.jpg</infon><infon key="id">F4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>19686</offset><text>A. The cofactor-binding cavity of M1.HpyAVI. Residues (yellow) that form direct hydrogen bonds with AdoMet (green) are indicated, distance of the hydrogen bond is marked. B. Superposition of AdoMet in the structures of M1.HpyAVI (green), DpnM (yellow) and M.TaqI (orange). The AdoMet terminal carboxyl of M.TaqI reveals different orientations. C. Cofactor binding affinity of wt-/mutants M1.HpyAVI proteins analyzed by microscale thermophoresis (MST). The binding affinity was determined between fluorescently labelled M1.HpyAVI protein and unlabeled AdoMet. The bound fraction is shown on the y-axis against the protein concentration. AdoMet (15 nM to 1 mM) was titrated into a fixed concentration of M1.HpyAVI wt/mutant proteins (800 nM). The dissociation constant (KD) is yielded according to the law of mass action from the isotherm derived of the raw data: M1.HpyAVI-wt: 41 ± 6 μM; M1.HpyAVI-D8A :212 ± 11 μM; M1.HpyAVI-D29A : 0 μM; M1.HpyAVI-H168A : 471 ± 51 μM; M1.HpyAVI-S198A : 242 ± 32 μM; M1.HpyAVI-T200A : 252 ± 28 μM; M1.HpyAVI-E216A : 0 μM. Standard for three replicates is indicated. Measurements were made with 40% LED and 40% laser power at 25°C. D. DNA methyltransferase activity of wide type protein and the mutants is quantified using radioactive assay. [3H]-methyl transferred to duplex DNA containing 5′-GAGG-3′ was quantified by Beckman LS6500 for 10 min, experiments were repeated for three times and data were corrected by subtraction of the background. E. Superposition of M1.HpyAVI (green) with M.MboIIA (cyan) and M.RsrI (magenta). Residues D29 and E216 are conserved through all the DNA MTases mentioned in Figure 3 (not shown in Figure 4).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21373</offset><text>To confirm the key residues for ligand binding, we prepared a series of single mutants by replacing D8, D29, H168, S198, T200, E216 with alanine and investigated their ligand binding affinity using microscale thermophoresis (MST) assay. As shown in Figure 4C, by contrast to the wild type enzyme, most mutants displayed variable reduction of KD value, among them the D29A and E216A mutants displayed no protein-AdoMet affinity at all.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>21808</offset><text>The results suggested that the hydrogen bonds formed by D29 and E216 with AdoMet were most crucial interactions for cofactor binding. Mutation of the two residues may directly prevent the methyl transfer reaction of M1.HpyAVI. The importance of D29 is preserved because it belongs to the catalytic active site DPPY, but the residue E216 has not been fully investigated even being a conserved amino acid throughout MTases (Figure 4E). E216 is the last residue of β2, which contacts the two hydroxyls of the ribose of AdoMet. Replacement of this residue by alanine completely abolishes the key hydrogen bonds for AdoMet-binding, and very likely blocks the methyl transfer reaction. To confirm this notion, [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the mutants. As shown in Figure 4D, the result of radiological assay agreed well with the MST measurement. The D29A and E216A mutants showed little or no methyl transfer activity, while other mutants exhibited reduced methyltransferase activity.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22847</offset><text>As mentioned previously, FXGXG is a conserved AdoMet-binding motif of DNA MTases. We also made mutants of “FMGSG” to alanine for every amino acid, and found that the F195A mutant was insoluble probably due to decreasing the local hydrophobicity upon this mutation. We subsequently investigated the ligand binding affinity and methyl transfer reaction of the other mutants using MST and a radiological assay. We found that G197 played a crucial role in AdoMet-binding, while mutagenesis of M196 and G199 did not influence cofactor binding and catalytic activity (Figure S2A and B). G197 is a conserved residue throughout the DNA MTases, and replacing by alanine at this site likely change the local conformation of cofactor-binding pocket. Mutagenesis on this glycine residue in M.EcoKI or M.EcoP15I also abolished the AdoMet-binding activity. Although mutational study could not tell the role of F195 in ligand binding due to the insolubility of the F195A mutant, structural analysis suggested the importance of this residue in AdoMet-binding. The phenyl ring of F195 forms a perpendicular π-stacking interaction with the purine ring of AdoMet, which stabilizes the orientation of AdoMet bound in the pocket of M1.HpyAVI (Figure S2C). In a separate scenario, mutagenesis of this residue in M.EcoRV has been proven to play an important role in AdoMet binding.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>24211</offset><text>Potential DNA-binding sites</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24239</offset><text>The putative DNA binding region of M1.HpyAVI involves the hairpin loop (residue 101-133), the TRD (residues 136-166), and a highly flexible loop (residues 33-58). The hairpin loop between β6 and β7 strands that carries a conserved HRRY sequence signature in the middle is proposed to insert into the minor groove of the bound DNA. As aforementioned, the TRD of M1.HpyAVI shows striking difference from the other DNA MTases, and the relaxed specificity of substrate recognition may be at least partially attributable to the disordered TRD.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24784</offset><text>In addition, the highly flexible loop immediately following the DPPY motif in M1.HpyAVI was poorly defined in electron density, exactly like the corresponding loops in the AdoMet-bound structures of M.PvuII, DpnM or M.TaqI that were invisible either. This loop, however, was largely stabilized upon DNA binding, as observed in the protein-DNA complex structures of M.TaqI (PDB ID 2IBS), M.HhaI (PDB ID 1MHT) and M.HaeIII (PDB ID 1DCT). The well-ordered loop in those structures directly contacts the flipping adenine and forms hydrogen bond with neighboring bases. These observations implied that the corresponding loop in other MTases, e.g. M1.HpyAVI, is likely responsible for reducing sequence recognition specificity and thus plays crucial roles in catalysis.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>25548</offset><text>Key residue for wider spectrum of substrate recognition</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>25604</offset><text>Previous research suggested that M1.HpyAVI from strain 26695 was the first N6 adenine MTase that can methylate the adenine of 5′-GAGG-3′/5′-GGAG-3′ or both two adenines of 5′-GAAG-3′, compared with the homologs from other strains that can methylate only one adenine of 5′-GAGG-3′. To answer why M1.HpyAVI displayed a wider specificity for DNA recognition, we randomly choose fifty of M1.HpyAVI sequences from hundreds of H. pylori strains for multiple sequence alignment. Based on sequence comparison and structural analysis, four residues including P41, N111, K165 and T166 were selected and replaced by serine, threonine, threonine and valine, respectively (Figure 5A). Then, a [3H]AdoMet radiological assay was applied to quantify the methyl transfer activity of the wide type protein and the mutants. As shown in Figure 5, when the substrate DNA contains 5′-GAGG-3′ or 5′-GAAG-3′, all the mutants showed no apparent difference of methyl transfer activity compared to the wt-M1.HpyAVI; but when the recognition sequence was 5′-GGAG-3′, the methyl transfer activity of the P41S mutant was significantly reduced compared to the wild type M1.HpyAVI.</text></passage><passage><infon key="file">oncotarget-07-40965-g005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>26782</offset><text>Sequence alignment, structural analysis and radioactive methyl transfer activity define the key residue for wider substrate specificity of M1.HpyAVI</text></passage><passage><infon key="file">oncotarget-07-40965-g005.jpg</infon><infon key="id">F5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>26931</offset><text>A. Sequence alignment of M1.HpyAVI from 50 H. pylori strains including 26695 revealed several variant residues. Residues P41, N111, K165 and T166 of M1.HpyAVI from strain 26695 were chosen based on structural analysis and sequence alignment (shown in red arrow). Amino-acid conservation is depicted using WebLogo (Crooks et al, 2004). B., C., D. Methyl transfer reactions were performed using wt-M1.HpyAVI, M1.HpyAVI-P41S, M1.HpyAVI-N111T, and M1.HpyAVI-K165R T166V, respectively. Radioactivity incorporated into the duplex DNA containing 5′-GAGG-3′, 5′-GAAG-3′ or 5′-GGAG-3′ was quantified by Beckman LS6500 for 10 min. The experiments were repeated for three times and data were corrected by subtraction of the background.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>27668</offset><text>Our experimental data identified P41 as a key residue determining the recognition of GGAG of M1.HpyAVI. This amino acid locates in the highly flexible loop between residues 33 and 58, which is involved in DNA binding and substrate recognition as shown above. Replacement by serine at this position definitely changes the local conformation and hydrophobicity, and probably some structural properties of the whole loop, which may in turn result in reduced specificity for sequence recognition of the enzyme from strain 26695.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>28193</offset><text>DISCUSSION</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28204</offset><text>Although the DNA-bound structure of previous investigation on a γ-class N6-adenine MTase revealed that the target adenine was rotated out of DNA helix, details of the methyl transfer process were still unclear. Additionally, recent studies reported the importance of N6-methyladenine in some eukaryotic species, but until now there has not been any N6-adenine MTases being identified in eukaryotes. Biochemical and structural characterization of M1.HpyAVI provides a new model for uncovering the methyl transfer mechanism and for investigating the N6-methyladenine in eukaryotes.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28788</offset><text>Oligomeric state of DNA MTases was long accepted as monomer, but our study indicated here that M1.HpyAVI exists as a dimer both in crystal and solution. Interestingly, some other β-class DNA exocyclic MTases showed similar oligomeric state in crystal and in solution, indicating that dimer may be the functional state shared by a subgroup of DNA MTases.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29145</offset><text>The highly flexible region (residues 33-58) and TRD (residues 133-163) of M1.HpyAVI are supposed to interact with DNA at minor and major grooves, respectively. These two structural characteristics may account for the substrate promiscuity of this enzyme. And residue P41 might be a key residue partially determining the substrate spectrum of M1.HpyAVI.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29498</offset><text>The missing loop between residues 33 and 58 may need DNA binding so as to form a stable conformation, which is similar to the condition of M.TaqI. Crystallization of M1.HpyAVI-DNA complex warrants future investigations, with the purpose of revealing the mechanism behind the wider substrate specificity of this enzyme.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>29817</offset><text>DNA methylation plays an important role in bacterial pathogenicity. DNA adenine methylation was known to regulate the expression of some virulence genes in bacteria including H.pylori. Inhibitors of DNA adenine methylation may have a broad antimicrobial action by targeting DNA adenine methyltransferase. As an important biological modification, DNA methylation directly influences bacterial survival. Knockout of M1.HpyAVI largely prevents the growth of H. pylori. Importantly, H. pylori is involved in 90% of all gastric malignancies. Appropriate antibiotic regimens could successfully cure gastric diseases caused by H.pylori infection. However, eradication of H. pylori infection remains a big challenge for the significantly increasing prevalence of its resistance to antibiotics. The development of new drugs targeting adenine MTases such as M1.HpyAVI offers a new opportunity for inhibition of H. pylori infection. Residues that play crucial roles for catalytic activity like D29 or E216 may influence the H.pylori survival. Small molecules targeting these highly conserved residues are likely to emerge less drug resistance.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30950</offset><text>In summary, the structure of M1.HpyAVI is featured with a disordered TRD and a key residue P41that located in the putative DNA binding region that may associate with the wider substrate specificity. Residues D29 and E216 were identified to play a crucial role in cofactor binding. As the first crystal structure of N6-adenine MTase in H.pylori, this model may shed light on design of new antibiotics to interfere the growth and pathogenesis of H.pylori in human.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>31413</offset><text>MATERIALS AND METHODS</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>31435</offset><text>Protein expression and purification</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>31471</offset><text>The ORF encoding M1.HpyAVI was inserted into the expression plasmid pET22b (Novagen, Massachusetts, USA) to produce a recombinant protein containing a C-terminal His-tag. In order to produce soluble protein, a chaperone plasmid PG-KJE8 (TaKaRa, Dalian, China) was co-expressed with M1.HpyAVI. The recombinant protein was purified with a three-step chromatography protocol using a Ni-NTA affinity column, a HiLoad 16/60 Superdex 200 column and a mono-S HR 5/5 column (1ml) (GE Healthcare, Uppsala, Sweden). Mutants of M1.HpyAVI were generated using the Muta-direct Site-directed Mutagenesis kit (SBS Genetech, Beijing, China) and produced using the same protocol with wide type protein.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32157</offset><text>Crystallization and data collection</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32193</offset><text>Crystallization trials were carried out for both the AdoMet-free and AdoMet-bound proteins using the hanging drop vapor diffusion technique. Crystals used for diffraction data collection of the apoprotein were grown under the condition of 1.0 M Bis-Tris, pH 9.0, 1.4 M ammonium tartrate, and the optimal crystallization condition for AdoMet-bound protein was 1.0 M Bis-Tris, pH 6.0, 14% PEG2000, 0.2 M lithium sulfate. X-ray diffraction data were collected at 100 K on beamline BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) using an ADSC Quantum 315r CCD detector. All data were indexed, integrated and scaled using the XDS program.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>32841</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>32880</offset><text>The structure of ligand-free M1.HpyAVI was determined by means of molecular replacement using the M.MboIIA (PDB ID 1G60) as a search model. Automated structure determination using Phaser gave a solution showing four subunits sitting in the asymmetric unit. The model was refined using the COOT graphics package manually and phenix.refine. The AdoMet-bound structure was determined by means of molecular replacement using the refined model of the apoprotein, and refined in the same way. Statistics from the data collection and structure refinement are summarized in Table 1. All figures representing the M1.HpyAVI structures were generated using the molecular visualization program PyMol.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>33569</offset><text>Detection of protein dimerization</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33603</offset><text>The interface information of M1.HpyAVI free form and AdoMet-bound form structures were analyzed using the PDBePISA (Proteins, Interface, Structures and Assemblies) web server.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>33779</offset><text>The protein molecular weight was determined by gel filtration using a FPLC system coupled to a Superdex 75 HR 10 / 30 column. The sizing standard was calibrated using the gel filtration calibration kit LMW (GE Healthcare, Uppsala, Sweden).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34019</offset><text>Binding affinity quantification via microscale thermophoresis (MST)</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34087</offset><text>Microscale thermophoresis was performed using the NT115 nanotemper technologies. M1.HpyAVI-wt and M1.HpyAVI-mutant proteins were fluorescently labeled using the protein label kit according to manufacturer's protocol. Affinity measurements were performed by using MST buffer (0.05% Tween-20 added as final concentration). A solution of unlabeled AdoMet was serially diluted from 1 mM to 15 nM. Equal volume of 0.8 μM labeled protein was mixed with the AdoMet and loaded into the silica capillaries. This binding curve can directly be fitted with the nonlinear solution of the law of mass action, with the dissociation constant (KD) as a result. Measurement was performed at 25°C using 40% LED power and 40%IR-laser power. The dissociation constant was calculated using the Nano-temper Analysis software.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34892</offset><text>Radioactive methyltransferase analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34931</offset><text>Several different DNA duplexes containing single site of 5′- GAGG-3′, 5′- GAAG-3′ or 5′-GGAG-3′ were used as substrate for methyl transfer reaction (Table S2). 0.1 μM of enzyme and 2 μM of S-[methyl-3H] adenosly methionine (China Isotope and Radiation Corporation, Beijing, China) were incubated at 37°C for 5 min, and then 5 μM of DNA substrate was added to initiate the reaction. Aliquots (20 μl) were taken out at 4-min time intervals and quenched with 2 N HCl. Subsequently, DNA of the mixture was purified using a DNA purification column (TIANGEN, Beijing, China) and the scintillation counting of tritiated DNA was quantified by Beckman LS6500 for 10 min. The background radioactivity was determined by omitting the enzyme from the reaction solution. All the reactions were performed in triplicate.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>35753</offset><text>SUPPLEMENTARY FIGURES AND TABLES</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35786</offset><text>CONFLICTS OF INTEREST</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35808</offset><text>The authors declare that they have no conflicts of interest.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35869</offset><text>ACCESSION CODES</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>35885</offset><text>Atomic coordinates and structure factors for apo-M1.HpyAVI and cofactor-bound M1.HpyAVI have been deposited in the PDB, with accession codes 5HEK and 5HFJ 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<collection><source>PMC</source><date>20201222</date><key>pmc.key</key><document><id>5603727</id><infon key="license">CC BY</infon><passage><infon key="article-id_doi">10.1038/ncomms11032</infon><infon key="article-id_pii">ncomms11032</infon><infon key="article-id_pmc">5603727</infon><infon key="article-id_pmid">27010430</infon><infon key="elocation-id">11032</infon><infon key="license">This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/</infon><infon key="name_0">surname:Janowski;given-names:Robert</infon><infon key="name_1">surname:Heinz;given-names:Gitta A.</infon><infon key="name_10">surname:Niessing;given-names:Dierk</infon><infon key="name_11">surname:Heissmeyer;given-names:Vigo</infon><infon key="name_12">surname:Sattler;given-names:Michael</infon><infon key="name_2">surname:Schlundt;given-names:Andreas</infon><infon key="name_3">surname:Wommelsdorf;given-names:Nina</infon><infon key="name_4">surname:Brenner;given-names:Sven</infon><infon key="name_5">surname:Gruber;given-names:Andreas R.</infon><infon key="name_6">surname:Blank;given-names:Michael</infon><infon key="name_7">surname:Buch;given-names:Thorsten</infon><infon key="name_8">surname:Buhmann;given-names:Raymund</infon><infon key="name_9">surname:Zavolan;given-names:Mihaela</infon><infon key="section_type">TITLE</infon><infon key="type">front</infon><infon key="volume">7</infon><infon key="year">2016</infon><offset>0</offset><text>Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>77</offset><text>The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.</text></passage><passage><infon key="section_type">ABSTRACT</infon><infon key="type">abstract</infon><offset>1004</offset><text> Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>1306</offset><text>Posttranscriptional gene regulation is involved in a wide range of cellular functions and its critical importance has been described for many developmental and differentiation processes. Consistently, mutations of factors involved in posttranscriptional gene regulation pathways were found associated with a number of genetically inherited diseases. The Roquin protein is essential in T cells for the prevention of autoimmune disease. This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs).</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>2652</offset><text>We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>3652</offset><text>We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology.</text></passage><passage><infon key="section_type">INTRO</infon><infon key="type">paragraph</infon><offset>4622</offset><text>Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_1</infon><offset>5275</offset><text>Results</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>5283</offset><text>SELEX identifies novel RNA ligands of Roquin-1</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>5330</offset><text>We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. Enriched sequences were clustered into so-called patterns with highly homologous sequences. Hereby, the algorithm at first identified frequent motifs of five to eight nucleotides length and subsequently used iterative cycles of proto-pattern formation to cluster sequences bearing two of such frequent motifs. A final aptamer pattern was built up by sequences bearing two frequent motifs and, at the same time, having high similarities also in other sequence parts. Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). Notably, our SELEX approach did not reveal the previously identified CDE sequence. We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>8579</offset><text>NMR analysis of Roquin-bound SL RNAs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>8616</offset><text>We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>10018</offset><text>Structures of ROQ bound to ADE SL RNAs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>10057</offset><text>To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>11492</offset><text>The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>13852</offset><text>NMR analysis of ROQ interactions with ADE SLs</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>13898</offset><text>We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>15076</offset><text>Mutational analysis of the ROQ-ADE interaction</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>15123</offset><text>To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>16950</offset><text>To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). The observed partial rescue may relate to the low, close to endogenous expression of these constructs (Supplementary Fig. 5b). However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>18696</offset><text>We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>20620</offset><text>Roquin binding to different SLs in the Ox40 3′-UTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>20673</offset><text>We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR.</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">title_2</infon><offset>22420</offset><text>Regulation of Ox40 expression via two motifs in its 3′-UTR</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>22481</offset><text>To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c).</text></passage><passage><infon key="section_type">RESULTS</infon><infon key="type">paragraph</infon><offset>24230</offset><text>To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">title_1</infon><offset>25165</offset><text>Discussion</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25176</offset><text>Recent structural and functional studies have provided first insight into the RNA binding of Roquin. Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. Some evidence for this is provided by a recent study by Landthaler and colleagues.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>25783</offset><text>Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>27734</offset><text>Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. It remains to be seen which exact features govern the recognition of the A20 SL by Roquin.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>28712</offset><text>The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c).</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>30235</offset><text>Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. Both were required for Regnase-1-mediated repression. These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31199</offset><text>Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence.</text></passage><passage><infon key="section_type">DISCUSS</infon><infon key="type">paragraph</infon><offset>31958</offset><text>The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>34078</offset><text>Methods</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>34086</offset><text>SELEX experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>34104</offset><text>Selection of Roquin-1-bound RNAs from a random RNA library was performed in three rounds of selection with increased stringency of washing (3 × 100 μl, 4 × 100 μl and 5 × 100 μl washing steps) and with decreased protein concentrations (250, 150 and 50 nM). Before selection, 100 μg recombinant Roquin-1 and Roquin-1 M199R N-terminal protein (residues 2–440) were biotinylated: proteins were incubated for 30 min on ice with 10 × molar excess of EZ-link PEG4-NHS-Biotin (Pierce) in PBS (0.1 mg ml−1). Subsequently, the biotinylated protein was purified via gel filtration (MicroSpin column P6, BioRad) and the loss of protein during the biotinylation procedure was estimated by SDS–PAGE and Coomassie staining. The efficiency of the biotinylation reaction was evaluated after spotting of unlabelled and labelled proteins onto a nitrocellulose membrane. After blocking the membrane with 1% BSA in PBS, it was incubated in streptavidin–PE (R-Phycoerythrin) diluted 1:1,000 in PBS for 30 min at room temperature (RT). Subsequently, the membrane was washed three times with PBS and fluorescence intensity of PE bound to biotinylated protein was determined by fluoroimaging (Raytest, FLA5000, 473 nm, Y510 filter).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>35349</offset><text>The RNA startpool containing the 47-nt random sequence as well as the RNA pools for the second and third selection rounds were transcribed in vitro from double-stranded PCR DNA, and protein-bound RNA was isolated and reverse transcribed before PCR amplification, as previously described. Following transcription, the samples were separated on an 8% PAGE, the bands excised and RNA purified. Every round of selection started by combining the RNA pool (400 pmol) with biotinylated protein and incubating the mix for 30 min at 37 °C. Subsequently, binding buffer-equilibrated streptavidin-magnetic beads were added and incubated (10 min, 37 °C) to bind the protein–RNA complexes, followed by washes. By boiling the beads in 0.2 mM EDTA in water for 3 min, protein and RNA molecules were released. After removal of beads, the solution served as template for reverse transcription (One-Step RT-PCR Kit, Qiagen) and from the obtained complementary DNA the RNA pool of the next round of selection was transcribed. The cDNAs from every selection round (startpool, round 1, round 2 and round 3) were used for Index-PCRs to analyse the pool composition at every stage during selection. Comparable amounts of the PCR products were combined to one cDNA library and analysed by Solexa Illumina sequencing.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>36660</offset><text>Sequence motif and structural analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>36699</offset><text>To identify sequence motifs to which Roquin specifically binds, we counted the number of occurrences of each hexamer (46=4,096 motifs) in the sequences obtained by SELEX. We then generated a data set of randomized sequences of the same nucleotide composition as the SELEX-derived sequences, by permuting the SELEX-derived sequences with a custom script. Finally, we counted the number of occurrences of each hexamer in the set of randomized sequences and computed the log2 ratio of the number of occurrences of each motif in the real and randomized sequence sets. To identify a shared sequence motif in the SELEX patterns that showed the strongest enrichment in our selection experiments, the top 100 patterns were analysed with the Motif-based sequence analysis tool MEME (http://meme-suite.org) using the default settings. This analysis revealed three sequence motifs of which the first is shown in Fig. 1b. For the construction of sequence logos, we screened the obtained nucleotide sequences from SELEX replicate 1 and extracted the nucleotide sequences including the 7-nt flanking regions. Sequence logos were constructed with WebLogo 2.8.2 (http://weblogo.berkeley.edu/).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>37877</offset><text>For the Ox40 3′-UTR sequence alignment, we extracted Multiz alignments of 60 Vertebrates from the UCSC mouse GRCm38/mm10 assembly for the genomic region chr4:156,016,498–156,016,520. For each species contained in the alignment, we extracted genomic coordinates of the aligned sequence, extended the coordinates by 10 nt upstream and downstream, and retrieved the extended sequences from the corresponding genome assemblies. Finally, sequences were aligned with ClustalW 2.1 with standard settings and the alignment was visualized using Jalview.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>38428</offset><text>To evaluate the structural context the inferred motif is located in, we first appended to the nucleotide sequences obtained from the SELEX experiment the SELEX primers 5′-GGAGAGATGTGAACTT-3′ and 5′-AGTTTCGTGGATGCCAC-3′ to the 5′- and 3′-end, respectively. Next, we screened for sequences that contained the inferred motif and performed secondary structure prediction on those sequences with RNAfold from the ViennaRNA package version 1.8 with parameters '-p -d2'. Next, we used a custom Perl script to parse the base-pairing probability file generated by RNAfold and to calculate an average base-pair probability over all sequences that contained the inferred motif.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>39107</offset><text>Production of proteins</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>39130</offset><text>Cloning of expression vectors for Roquin-1 ROQ (residues 147–326), ROQ (residues 171–326) and Roquin-1 N-term (residues 2–440) was carried out by standard procedures as described previously. Briefly, PCR-amplified fragments were put into pETM11 and pETTrx1a vectors based on pET24d as provided by the Protein Expression and Purification Facility at Helmholtz Zentrum München. All vectors contained tobacco etch virus (TEV) protease recognition sites for subsequent proteolytic removal of the tags. All length-variable Roquin-1 expression constructs were designed and cloned via restriction sites NcoI (5′) and XhoI (3′). ROQ domain RNA-binding mutants were cloned by Quick change PCR with high-fidelity Phusion DNA polymerase and subsequent treatment with DnpI. Alternatively, we used conventional cloning with a two-step PCR protocol and enzymatic restriction.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40003</offset><text>The Roquin-1 fragments (147–326) and (171–326) were expressed as N-terminal His6-thioredoxin fusion proteins as recently described. Isotope-labelled protein for NMR studies was expressed in M9 minimal medium supplemented with 0.5 g l−1 15N ammonium chloride and 2 g l−1unlabelled or [U-13C] glucose. For the preparation of deuterated proteins, cells were adapted and grown as described previously. Briefly, we used a protocol with stepwise adaptation of cells to deuterium changing buffer from no D2O, low glucose to 50% D2O, low glucose and finally 99.5% D2O with deuterated glucose. The Roquin-1 N-terminal domain (residues 2–440) was expressed and purified essentially as described above for the ROQ domain, but no thioredoxin tag was used. For Roquin-1 N-terminal domain, all expression media and the final buffer contained 100 or 25 μM of zinc chloride, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>40897</offset><text>RNA preparation</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>40913</offset><text>RNAs were synthesized and purchased from IBA GmbH (Göttingen, Germany), purified via PAGE followed by two steps of desalting. No major impurities were seen in NMR spectra. Complex formation for crystallography and NMR experiments was achieved by dissolving the lyophilized RNA in water or NMR buffer. This stock solution was snap-cooled by boiling at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. All RNAs were stored at −80 °C, to avoid degradation and thermodynamically favoured duplex formation.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>41461</offset><text>Full length and fragments of Ox40 3′-UTR mRNA were produced by in vitro transcription (IVT) from DNA templates harbouring a T7 promoter site either with direct incorporation of α-32P-labelled UTP or subsequent 3′-labelling of purified RNA with γ-32P-labelled ATP. DNA templates were cloned by primer extension PCR. For IVT, 50–150 nM of DNA were incubated with 11 mM magnesium chloride, 8% (w/v) PEG8000, 1.25 mM of each NTP and 0.05 mg ml−1 of T7 polymerase in 1 × standard reaction buffer for 3–5 h at 37 °C. Labelled RNAs were produced in 50 μl reactions and purified via spin columns and directly subjected to EMSA assays. Unlabelled RNAs were produced in reactions of 500–5,000 μl. After IVT, the reactions were separated on 8% denaturing SDS–PAGEs, RNA of interest excised and eluted from the gel using the Elutrap kit (GE Healthcare). After elution, RNAs were dialysed against water and lyophilized. Subsequently, RNAs were dissolved in water and stocks generated by boiling them at 95 °C for 5 min and transferred to an ice-cold bath for 10 min before aliquoting. Labelling for EMSA assays was carried out as for short motifs and described recently. As a modification, dephosphorylation was performed for 30 min and 3′-phosphorylation with γ-32P-labelled ATP and T4 polynucleotide kinase for 90 min for higher efficiency, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>42857</offset><text>NMR spectroscopy</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>42874</offset><text>NMR measurements of Roquin-1 ROQ (147–326) and ROQ (171–326) were performed in buffers as described, mixed with 10% D2O. Backbone chemical shift assignments of ROQ (171–326) with 1.1- to 1.2-fold excess of the Ox40 ADE-like SL motif or consensus ADE SL RNAs were recorded with protein concentrations of 350–400 μM. HNCA, HNCACB, HNCO, HNcaCO and 3D 15N-edited NOESY spectra were acquired at 298K on Bruker Avance III spectrometers at field strengths corresponding to 600 and 800 MHz proton Larmor frequency, equipped with TCI cryogenic probe heads. Spectra of ROQ in complex with Ox40 CDE-like SL RNA and the RNA alone have been reported before. Spectra were processed with Topspin3.2 and analysed with CCPNMR Analysis and Sparky. For RNA motifs, one- and two-dimensional imino NOESY spectra with water-flip-back WATERGATE were recorded at 600–900 MHz, at 278 and 298 K at 150–350 μM RNA concentrations. Sequential assignments were guided by secondary structure predictions with mfold and supported by 15N chemical shifts from natural abundance SOFAST-HMQC experiments.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>43968</offset><text>Electrophoretic mobility shift assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>44006</offset><text>The EMSAs with ROQ domain and individual motifs were performed as described previously. In short, for the binding reaction a mastermix containing transfer RNA, 32P-labelled SL RNA and reaction buffer was prepared and then mixed with dilutions of the recombinant proteins to achieve the indicated protein concentrations. The binding was performed for 10 min at RT or 20 min on ice in 20 μl reaction volume in the presence of 2.5 μg μl−1 tRNA from baker’s yeast (Sigma), 500 pM 32P-labelled RNA, 20 mM HEPES (pH 7.4), 50 mM NaCl, 1 mM MgCl2, 1 mM dithiothreitol and 1 μg μl−1 BSA. For the binding reaction of Roquin-1 N-terminal with full-length Ox40 3′-UTRs or fragments thereof, ∼1 pmol of RNA was incubated with protein concentrations between 0 and 1,000 μM in a volume of 20 μl. RNP complexes were resolved by PAGE (6% polyacrylamide, 5% glycerol, 0.5 × TBE) at 120 V for 40 min at RT. Gels were then fixed, dried and exposed to a phosphor imager screen.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>45015</offset><text>X-ray crystallography</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>45037</offset><text>The crystallization experiments for ROQ–RNA complexes were performed at the X-ray Crystallography Platform at Helmholtz Zentrum München. The crystals of both, Roquin-1 ROQ (171–326) with Ox40 ADE-like SL motif (22mer, 5′-UCCACACCGUUCUAGGUGCUGG-3′) and with the consensus SELEX-derived ADE SL motif (20mer, 5′-UGACUGCGUUUUAGGAGUUA-3′) were obtained from the same condition: 100 mM Bis-Tris buffer pH 5.5, 200 mM sodium chloride and 25% (v/w) PEG 3350. Crystallization was performed using the sitting-drop vapour-diffusion method at 292 K in 24-well plates and a protein concentration of 12 mg ml−1. The crystals appeared after 1 day. For the X-ray diffraction experiments, the crystals of both co-complexes were mounted in a nylon fibre loop and flash cooled to 100 K in liquid nitrogen. The cryoprotection was performed for 2 s in reservoir solution complemented with 20% (v/v) ethylene glycol. Diffraction data for ROQ Ox40 ADE-like motif was collected on the ID29 beamline (ESRF, Grenoble, France) using a Pilatus 6M at a wavelength of 1.25363 Å. Diffraction data for the ROQ-ADE complex were collected using Pilatus 2M detector at 1.00003 Å wavelength at PXIII beamline at SLS (Villigen, Switzerland). All data sets were indexed and integrated using XDS and scaled using SCALA. Intensities were converted to structure–factor amplitudes using the programme TRUNCATE. Table 1 summarizes data collection and processing statistics for both data sets.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>46524</offset><text>Structure determination and refinement</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>46563</offset><text>The structure of both ROQ-Ox40 ADE-like SL and ROQ-ADE SL were solved by molecular replacement using the native Roquin-1 ROQ (147–326) structure as a search model (PDB: 4QI0 (ref.)). Model building was performed in COOT. RNA molecules were modelled manually. The refinement of both structures was done in REFMAC5 (ref.) using the maximum-likelihood target function including translation, libration and screw-rotation displacements of a pseudo-rigid body (TLS). For the ROQ-ADE SL structure, non-crystallographic symmetry (NCS) averaging was implemented. The final models are characterized by R and Rfree factors of 21.8 and 25.7% for ROQ-Ox40 ADE-like SL, and 18.6 and 23.4% for ROQ-ADE SL (Table 1), respecively. The stereochemical analysis of both final models was done in PROCHECK and MolProbity. It indicates that there are no residues with generously allowed or unfavourable backbone dihedral angles, and that 99.4% (for ROQ-Ox40 ADE-like SL structure) and 92.3% (for ROQ-ADE SL structure) of all residues are in the core region of the Ramachandran plot.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>47625</offset><text>Functional assays</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>47643</offset><text>Functional assays determining the Roquin-mediated regulation of Ox40 with different 3′-UTR variants were performed as described previously. In brief, Rc3h1/2−/− mouse embryonic fibroblast (MEF) cells, stably transduced with a doxycycline-inducible Roquin-1-p2A-mCherry construct, were retrovirally infected with Ox40 constructs of different 3′-UTR length or mutation, which led to the expression of Ox40 on the cell surface (CDE-like mutation changing nt 14–16 GCA to CGT, ADE-like mutation changing nt 15–17 from GGT to CCA). Forty-eight hours after infection, the cells were split and one half of the cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 and mCherry, connected via the self-cleaving peptide p2A. Thus, Roquin-expressing cells were marked by mCherry expression. Sixteen to 20 h after induction, the cells were harvested, stained with allophycocyanin (APC)-conjugated anti-Ox40 and analysed by flow cytometry. To compare the Ox40 expression levels achieved by different constructs, the relative Ox40 mean fluorescence intensity (MFI) was determined by dividing the MFI of treated (mCherry+) cells by the MFI of untreated (mCherry−) cells.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>48849</offset><text>Mouse experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>48867</offset><text>Compound mutant mice with the Rc3h1fl/fl (ref.) and Rc3h2fl/fl (ref.) (denoted Rc3h1/2fl/fl), as well as Cd4-Cre-ERT2 (ref.) and Gt(ROSA)26Sortm1(rtTA*M2)Jae alleles were maintained on a C57BL/6 genetic background. All animals were housed in a pathogen-free barrier facility in accordance with the Ludwig-Maximilians-University München institutional, state and federal guidelines.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49249</offset><text>Generation of overexpression vectors</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49286</offset><text>Expression constructs of Roquin-1 and Ox40 were cloned into a modified pRetroX-Tight vector (Clontech). The puromycine-resistance cassette was removed and a cassette containing attR1-ccdB-attR2 was inserted, to generate a Gateway destination vector. Roquin-1 and Ox40 constructs were inserted by LR reaction (Invitrogen). Any mutants thereof were generated by site-directed mutagenesis.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>49673</offset><text>Virus production</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>49690</offset><text>Replication-deficient retrovirus production and T-cell transduction was performed as previously described. Briefly, retroviral and packaging plasmids were introduced into HEK293T cells by calcium-phosphate transfection. Forty-eight hours after transfection, cell culture supernatants containing the retrovirus particles were harvested, passed through 0.45-μm filters and stored at −80 °C.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>50085</offset><text>Cell isolation and culture</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>50112</offset><text>Splenocytes were isolated from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice. CD4+ T cells were isolated by negative selection with magnetic beads according to the manufacturer’s instructions (Stem Cell Technologies). CD4+ T cells were cultured in DMEM medium supplemented with 10% (vol/vol) fetal bovine serum, 1 × nonessential amino acids (Lonza), 10 mM HEPES pH 7.4 (Invitrogen), 50 μM β-mercaptoethanol (Invitrogen) and 100 U ml−1 penicillin–streptomycin (Invitrogen). Rc3h1/2fl/fl deletion was induced by addition of 4′OH-Tamoxifen (0.3 μM) for 24 h. For TH1 differentiation, CD4+ T cells were cultured in six-well plates pre-coated with goat anti-hamster IgG (MP Biochemicals) and DMEM medium further supplemented with anti-CD3 (0,25 μg ml−1), anti-CD28 (2,5 μg ml−1), IL-12 (10 ng ml−1) and anti-IL-4 (10 μg ml−1) for 40 h. Cells were then infected with retroviral constructs, allowing reconstitution with either Roquin-1, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A, and cultured in IL-2 containing media (20 U ml−1). Forty-eight hours after transduction, the cells were split and one half of cells was treated with doxycycline (1 μg ml−1), to induce expression of Roquin-1 WT and Roquin-1 mutants. Twenty-four hours after induction, the cells were harvested for analysis by immunoblot and flow cytometry with the indicated antibodies (1:200 anti-mouse Icos-biotin clone 7E–17G9 (eBioscience); 1:200 Streptavidin-PerCP (Becton Dickinson); 1:200 anti-mouse Ox40-PE clone OX-86 (eBioscience)).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>51678</offset><text>Immunoblot analysis</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>51698</offset><text>CD4+ T cells were incubated for 15 min on ice with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% (vol/vol) Nonidet-P40, 1.5 mM MgCl2 and protease inhibitor mix without EDTA (Roche) and 1 mM dithiothreitol). Lysate was cleared by centrifugation (10 min, 10 000 g, 4 °C). Immunoblotting was performed by standard protocols with hybridoma supernatants containing monoclonal antibody recognizing Roquin-1 and Roquin-2 (anti-roquin, clone 3F12).</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>52168</offset><text>mRNA decay experiments</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>52191</offset><text>Hela Tet-Off Advanced Cells (Clontech 631156) were stably transduced with retroviruses expressing different Ox40 constructs. FACS analysis 41 h post transduction revealed similar Ox40 surface expression levels on all five cell samples. After transduction, the cell lines were initially cultured for at least 48 h without doxycycline, to ensure high Ox40-expression levels. For each time point, 400 000 cells were spread on one well in a six-well plate. To switch off Ox40-transcription, doxycycline was supplied with the medium at time point 0. After one washing step with PBS, cells were directly harvested from each well with Trizol before Dox application (0 h), as well as 2, 3 and 4 h after Dox application. RNA was isolated using standard Trizol protocols. Reverse transcription was performed with the Qiagen Quantitect Reverse Transcription Kit following the manufacturer’s protocols. Quantitative PCR was carried out on a Roche Light Cycler 480 using the Light Cycler 480 Probes Master Mix and primer-/probe-combinations from Roches Universal Probe Library. Relative mRNA expression levels were calculated by normalization to the housekeeper gene ywhaz.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_2</infon><offset>53364</offset><text>Surface plasmon resonance</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>53390</offset><text>ROQ–RNA binding experiments were performed on a BIACORE 3000 instrument (Biacore Inc.). ROQ domain was diluted to a final concentration of 35 μg ml−1 in 10 mM HEPES pH 7.0 and chemically immobilized (amine coupling) onto CM5 sensor chips (Biacore Inc.). The RNA samples were diluted in the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM MgCl2 and 0.005% Tween 20) to the final concentration of 31.25, 62.5, 125, 250 and 500 nM, and 1 and 2 μM, and injected over the sensor chip surface at 30 μl min−1 at 10 °C. The RNA samples were injected onto the sensor chip from the lowest to the highest concentration. Each RNA-type sample was tested three times with the exception of Mut1–3 two times. Injection of 250 nM RNA was always performed in duplicate within each experiment. To subtract any background noise from each data set, all samples were also run over an unmodified sensor chip surface. Data were analysed using BIAevaluation programme (Biacore Inc.) (Supplementary Fig. 7). For each measurement, the equilibrium dissociation constant was calculated (KD) from steady state binding. The KD from three independent experiments were used to calculate the mean values of these variables and the s.e.m. The results for all tested RNA samples are compared in Table 2.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">title_1</infon><offset>54700</offset><text>Additional information</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>54723</offset><text>Accession codes: Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5F5H and 5F5F for the ROQ-Ox40 ADE-like SL and ROQ-ADE SL, respectively. Chemical shifts of the ROQ-Ox40 ADE-like SL and ROQ-ADE SL have been deposited in the Biological Magnetic Resonance Data Bank under accession codes 26587 and 26588, respectively.</text></passage><passage><infon key="section_type">METHODS</infon><infon key="type">paragraph</infon><offset>55100</offset><text>How to cite this article: Janowski, R. et al. Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40. Nat. Commun. 7:11032 doi: 10.1038/ncomms11032 (2016).</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">title_1</infon><offset>55278</offset><text>Supplementary Material</text></passage><passage><infon key="fpage">160</infon><infon key="lpage">165</infon><infon key="name_0">surname:Turner;given-names:M.</infon><infon key="name_1">surname:Hodson;given-names:D.</infon><infon key="pub-id_pmid">22326859</infon><infon key="section_type">REF</infon><infon key="source">Curr. Opin. 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Immunol.</infon><infon key="type">ref</infon><infon key="volume">11</infon><infon key="year">2010</infon><offset>58853</offset><text>Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>58985</offset><text>The authors declare no competing financial interests.</text></passage><passage><infon key="section_type">SUPPL</infon><infon key="type">footnote</infon><offset>59039</offset><text>Author contributions A.S. carried out cloning, protein expression and purification, and NMR experiments. R.J. performed crystallization and structure determination. G.A.H. carried out EMSA assays and SELEX experiments, and functional studies were performed by G.A.H, N.W. and S.B. M.B. and R.B. helped setting up the SELEX experiments and identified patterns from NGS data, on the basis of which A.G. and M.Z. analysed the motifs and secondary structures. T.B. contributed unpublished reagents and advice. A.S., G.A.H., R.J., V.H., D.N. and M.S. designed the project and wrote the paper. All authors discussed the results and commented on the manuscript.</text></passage><passage><infon key="file">ncomms11032-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>59694</offset><text>SELEX identifies a novel SL RNA ligand of Roquin-1.</text></passage><passage><infon key="file">ncomms11032-f1.jpg</infon><infon key="id">f1</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>59746</offset><text>(a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. The labels correspond to the versions of the genome assemblies in the UCSC server (see Method section). rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. See also Supplementary Fig. 1.</text></passage><passage><infon key="file">ncomms11032-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>60600</offset><text>NMR analysis of the SL RNAs used in this study.</text></passage><passage><infon key="file">ncomms11032-f2.jpg</infon><infon key="id">f2</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>60648</offset><text>Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). The respective SL RNAs and their base pairs are indicated. Red asterisks indicate NMR signals of the protein. Black asterisks in a indicate a second conformation (see Supplementary Notes). Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. Red nucleotides indicate significant chemical shift changes observed. The dotted green line between G6 and G15 in a highlights a G–G base pair.</text></passage><passage><infon key="file">ncomms11032-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>61352</offset><text>Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA.</text></passage><passage><infon key="file">ncomms11032-f3.jpg</infon><infon key="id">f3</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>61417</offset><text>(a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). Selected RNA bases and protein secondary structure elements are labelled. (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). Only RNA-interacting residues that are different in both structures are shown. Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). The protein chain is shown in turquoise and the RNA is shown in grey. Atoms are colour coded according to charge. (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. Lys259 interacts with the phosphate groups of U10 and U11. (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. In d – f, amino acids are shown in turquoise and blue, nucleotides in grey colour. See also Supplementary Notes and Supplementary Fig. 2.</text></passage><passage><infon key="file">ncomms11032-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63093</offset><text>NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA.</text></passage><passage><infon key="file">ncomms11032-f4.jpg</infon><infon key="id">f4</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63170</offset><text>(a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). Selected shifts of amide resonances are indicated. (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. Gaps indicate prolines. (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). See also Supplementary Notes and Supplementary Fig. 3.</text></passage><passage><infon key="file">ncomms11032-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>63754</offset><text>Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR.</text></passage><passage><infon key="file">ncomms11032-f5.jpg</infon><infon key="id">f5</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>63840</offset><text>(a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5).</text></passage><passage><infon key="file">ncomms11032-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_title_caption</infon><offset>64624</offset><text>Functional importance of Roquin-1 target motifs in cells.</text></passage><passage><infon key="file">ncomms11032-f6.jpg</infon><infon key="id">f6</infon><infon key="section_type">FIG</infon><infon key="type">fig_caption</infon><offset>64682</offset><text>(a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). Arrows indicate the individual binding events to either motif. It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). Error bars represent the mean of technical duplicates in one experiment. mRNA half-life times were calculated with Graph Pad Prism. Data are representative of two experiments with similar results.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>66346</offset><text>Data collection and refinement statistics.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table</infon><infon key="xml"><?xml version="1.0" encoding="UTF-8"?>
<table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"> </th><th align="center" valign="top" charoff="50"><bold>ROQ-</bold><italic><bold>Ox40</bold></italic>
<bold>ADE-like SL</bold></th><th align="center" valign="top" charoff="50"><bold>ROQ-ADE SL</bold></th></tr></thead><tbody valign="top"><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Data collection</italic></td></tr><tr><td align="left" valign="top" charoff="50"> space group</td><td align="center" valign="top" charoff="50"><italic>P</italic>2<sub>1</sub>2<sub>1</sub>2</td><td align="center" valign="top" charoff="50"><italic>P</italic>2<sub>1</sub>2<sub>1</sub>2<sub>1</sub></td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> Cell dimensions</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>a</italic>, <italic>b</italic>, <italic>c</italic> (Å)</td><td align="center" valign="top" charoff="50">89.66, 115.79, 42.61</td><td align="center" valign="top" charoff="50">72.90, 89.30, 144.70</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>α, β, γ</italic> (°)</td><td align="center" valign="top" charoff="50">90, 90, 90</td><td align="center" valign="top" charoff="50">90, 90, 90</td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">50–2.23 (2.29–2.23)</td><td align="center" valign="top" charoff="50">50–3.0 (3.08–3.00)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>merge</sub></td><td align="center" valign="top" charoff="50">5.9 (68.3)</td><td align="center" valign="top" charoff="50">14.8 (93.8)</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>I</italic>/σ<italic>I</italic></td><td align="center" valign="top" charoff="50">14.9 (2.1)</td><td align="center" valign="top" charoff="50">16.7 (3.1)</td></tr><tr><td align="left" valign="top" charoff="50"> Completeness (%)</td><td align="center" valign="top" charoff="50">98.7 (97.7)</td><td align="center" valign="top" charoff="50">99.9 (99.9)</td></tr><tr><td align="left" valign="top" charoff="50"> Redundancy</td><td align="center" valign="top" charoff="50">3.9 (3.7)</td><td align="center" valign="top" charoff="50">13.2 (12.7)</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Resolution (Å)</td><td align="center" valign="top" charoff="50">2.23</td><td align="center" valign="top" charoff="50">3.00</td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections</td><td align="center" valign="top" charoff="50">21,018</td><td align="center" valign="top" charoff="50">18,598</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic><sub>work</sub>/<italic>R</italic><sub>free</sub></td><td align="center" valign="top" charoff="50">21.8/25.7</td><td align="center" valign="top" charoff="50">18.6/23.4</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"> No. atoms</td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">2,404</td><td align="center" valign="top" charoff="50">4,820</td></tr><tr><td align="left" valign="top" charoff="50"> Ligand/ion</td><td align="center" valign="top" charoff="50">894</td><td align="center" valign="top" charoff="50">1,708</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="center" valign="top" charoff="50">99</td><td align="center" valign="top" charoff="50">49</td></tr><tr><td align="left" valign="top" charoff="50"><italic> B</italic>-factor overall</td><td align="center" valign="top" charoff="50">47.2</td><td align="center" valign="top" charoff="50">60.4</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50"><italic>Root mean squared deviations</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="center" valign="top" charoff="50">0.006</td><td align="center" valign="top" charoff="50">0.014</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="center" valign="top" charoff="50">1.07</td><td align="center" valign="top" charoff="50">1.77</td></tr><tr><td align="left" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td><td align="center" valign="top" charoff="50"> </td></tr><tr><td colspan="3" align="center" valign="top" charoff="50">Ramachandran plot</td></tr><tr><td align="left" valign="top" charoff="50"> Most favoured (%)</td><td align="center" valign="top" charoff="50">98.6</td><td align="center" valign="top" charoff="50">99.8</td></tr><tr><td align="left" valign="top" charoff="50"> Additional allowed (%)</td><td align="center" valign="top" charoff="50">1.4</td><td align="center" valign="top" charoff="50">0.2</td></tr></tbody></table>
</infon><offset>66389</offset><text> ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection space group P21212 P212121 Cell dimensions a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70 α, β, γ (°) 90, 90, 90 90, 90, 90 Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00) Rmerge 5.9 (68.3) 14.8 (93.8) I/σI 14.9 (2.1) 16.7 (3.1) Completeness (%) 98.7 (97.7) 99.9 (99.9) Redundancy 3.9 (3.7) 13.2 (12.7) Refinement Resolution (Å) 2.23 3.00 No. reflections 21,018 18,598 Rwork/Rfree 21.8/25.7 18.6/23.4 No. atoms Protein 2,404 4,820 Ligand/ion 894 1,708 Water 99 49 B-factor overall 47.2 60.4 Root mean squared deviations Bond lengths (Å) 0.006 0.014 Bond angles (°) 1.07 1.77 Ramachandran plot Most favoured (%) 98.6 99.8 Additional allowed (%) 1.4 0.2 </text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67293</offset><text>ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67373</offset><text>For each data set, only one crystal has been used.</text></passage><passage><infon key="file">t1.xml</infon><infon key="id">t1</infon><infon key="section_type">TABLE</infon><infon key="type">table_footnote</infon><offset>67424</offset><text>*Values in parentheses are for highest-resolution shell.</text></passage><passage><infon key="file"></infon><infon key="id">t2</infon><infon key="section_type">TABLE</infon><infon key="type">table_title_caption</infon><offset>67481</offset><text>KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1.</text></passage></document></collection>
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