s2fieldsofstudy: Chemistry Biology

text:
Interactions of the periplasmic binding protein CeuE with Fe(III) n-LICAM4− siderophore analogues of varied linker length

Bacteria use siderophores to mediate the transport of essential Fe(III) into the cell. In Campylobacter jejuni the periplasmic binding protein CeuE, an integral part of the Fe(III) transport system, has adapted to bind tetradentate siderophores using a His and a Tyr side chain to complete the Fe(III) coordination. A series of tetradentate siderophore mimics was synthesized in which the length of the linker between the two iron-binding catecholamide units was increased from four carbon atoms (4-LICAM4−) to five, six and eight (5-, 6-, 8-LICAM4−, respectively). Co-crystal structures with CeuE showed that the inter-planar angles between the iron-binding catecholamide units in the 5-, 6- and 8-LICAM4− structures are very similar (111°, 110° and 110°) and allow for an optimum fit into the binding pocket of CeuE, the inter-planar angle in the structure of 4-LICAM4− is significantly smaller (97°) due to restrictions imposed by the shorter linker. Accordingly, the protein-binding affinity was found to be slightly higher for 5- compared to 4-LICAM4− but decreases for 6- and 8-LICAM4−. The optimum linker length of five matches that present in natural siderophores such as enterobactin and azotochelin. Site-directed mutagenesis was used to investigate the relative importance of the Fe(III)-coordinating residues H227 and Y288.

Scientific RepoRts | 7:45941 | DOI: 10.1038/srep45941 In Gram-negative bacteria, Fe(III)-siderophore complexes are transported into the periplasm, where they are captured by specific PBPs 16 . The PBP then delivers the Fe(III)-siderophore complexes to a transporter in the inner membrane for delivery into the cytoplasm, where the Fe(III) is released 6 . The crystal structures of a number of siderophore PBPs have been determined, and while their sequence identity can be as low as 15%, they are structurally similar, being comprised of two domains connected through a long α -helical backbone 16 .
C. jejuni does not itself synthesize siderophores, but capitalizes on their secretion by other bacteria, such as E. coli. CeuE, an integral part of the CeuBCDE ABC transporter of C. jejuni, is responsible for capturing Fe(III)-siderophore complexes in the periplasm for delivery to their respective inner membrane transporter. It is not surprising that, as a scavenger, C. jejuni can utilize a diverse range of catecholate siderophores for the uptake of Fe(III), including tetradentate siderophores such as azotochelin and the enterobactin-derived bis(2,3-dihydroxybenzoyl-L-Ser) (Fig. 1) 17 . Indeed, the periplasmic enterobactin esterase Cee has been shown to play an important role in the uptake of Fe(III) in C. jejuni, since it hydrolyzes the enterobactin backbone in the periplasm followed by the import of Fe(III)-enterobactin fragment complexes, including bis (2,3-dihydroxybenzoyl-L-Ser), into the cytoplasm 18 . The observation that there is no known cytoplasmic enterobactin esterase in C. jejuni suggests that hexadentate analogues may first require cleavage to their tetradentate counterparts before transportation into the cytoplasm 18 .
Previously we cloned and overexpressed CeuE 1 , synthesized two catecholate siderophore analogues and probed their interaction with the protein using a combination of solution and structural approaches 19,20 . The first complex was with MECAM 6− (PDB ID: 2CHU) 19 , a synthetic enterobactin analogue that uses three catecholate arms to chelate Fe(III). However, since studies showed that tetradentate bis-catecholates can also act as siderophores for other bacterial species 21 , we subsequently synthesized the tetradentate analogue 4-LICAM 4− , which contains a four-atom linker between the two catecholamide units (Fig. 1). Co-crystallization of its Fe(III) complex with CeuE revealed the formation of a 1:1:1 complex CeuE-[Fe III (4-LICAM)] − (PDB ID: 5A1J 20 ). The octahedral coordination of the Fe(III) ion was completed by two protein residues, Tyr288 and His227. Subsequent studies 22 showed that 4-LICAM 4− was a good mimic of the natural tetradentate linear enterobactin hydrolysis product: bis(2,3-dihydroxybenzoyl-L-Ser) (PDB ID: 5ADV, 5ADW), (Fig. 1). Four homologues of CeuE identified using the Dali server (PDB ID: 3GFV, 4JCC, 3TEF and 4MX8) with Z-scores greater than 30, were all shown to contain the conserved Tyr and His residues, suggesting that this mode of binding is conserved in these bacteria 23 .
Here we report a combined solution and structural study of a series of synthetic tetradentate analogues of the enterobactin-derived bis(2, 3-dihydroxybenzoyl-L-Ser), to explore the ligand-binding promiscuity of CeuE, both wild type and with mutations of the Fe(III) binding residues H227 and Y288. The length of the linker between the Fe(III)-binding catecholamide units was increased from four carbon atoms (4-LICAM 4− ) to five, six and eight (5-, 6-, 8-LICAM 4− , respectively). Site directed mutagenesis was used to probe the relative contribution of H227 and Y288 to ligand binding.
Fe(III) complexes of 5-LICAM 4− , 6-LICAM 4− and 8-LICAM 4− were co-crystallized with CeuE and their crystal structures determined. The geometrical parameters obtained and those previously published were then correlated with the dissociation constants of the four synthetic [Fe III (n-LICAM)] − complexes, with n = 4, 5, 6 or 8, from CeuE, as measured by intrinsic fluorescence quenching. Analogous studies were carried out with CeuE mutants in which His227 and Tyr288 were replaced with non-coordinating residues.

Results and Discussion
The binding of all the n-LICAM ligands was first measured for wild type CeuE. To probe the relative contributions of His227 and Tyr288 to Fe(III)-siderophore binding, site-directed mutagenesis was then used to replace the side chains of the two Fe(III)-coordinating amino acids with non-coordinating side chains of similar size. His227 was mutated to either leucine (H227L) or alanine (H227A), while Y288 was mutated to phenylalanine (Y288F). In addition, the double mutants H227L/Y288F and H227A/Y288F were created.
Crystal structures of the wild type ligand complexes. Overall fold. For brevity, the four [Fe III (n-LICAM)] − complexes will henceforth be referred to as Fe-4-Lic, Fe-5-Lic, Fe-6-Lic or Fe-8-Lic and the four CeuE-[Fe III (n-LICAM)] − complexes as CFe-4-Lic, CFe-5-Lic, CFe-6-Lic or CFe-8-Lic. The overall fold of C. jejuni CeuE has been described in detail previously 19,20 , and the structure of CFe-4-Lic has also been reported 19,20 . The ligand lies in a cleft between the two domains of the protein, shown for CFe-5-Lic in Fig. 2a and b. In the three co-crystal structures, CFe-5-Lic, CFe-6-Lic and CFe-8-Lic, there is well defined density for the whole protein chain and the overall fold is very similar to that in CFe The siderophore-binding pocket. There is well-defined electron density for the Fe(III) cation and the n-Lic ligand in all three complexes, as shown in Fig. 3(a-c). The linkers between the two catecholate binding units are well ordered, with B-values close to the average value for each structure, in spite of the linker pointing toward the solvent with essentially no contacts to the protein (Fig. 2). The longer linker of 8-Lic is notably well ordered considering its length and inherent potential for flexibility. This tolerance of longer linkers can be rationalized by the lack of protein contacts observed for the linker region in the structures. As in the CFe-4-Lic structure, in the three new complexes the octahedral coordination sphere of the iron centers is composed of four oxygen donors from the two catecholate groups with the two remaining donor atoms being provided by the side chains of His227 and Tyr288, Fig. 3(d-f) 20 . The residues that contribute to the siderophore complex binding are depicted in Fig. 3(e) for CFe-5-Lic. Three arginine residues, Arg118, Arg205 and Arg249, interact with the catechol oxygen atoms via hydrogen bonding. In addition, there are hydrogen bonds between Lys121 and a catechol oxygen, and Gln98 and an amide carbonyl. Other than Gln98, only water is involved in hydrogen bonding to the linker region.
Superposition of the four structures, Fig. 3(f), reveals subtle differences in the binding mode. The accommodation of the four, five, six or eight carbons in the linker is seen at the top of Fig. 3(f). The catecholate unit shown on the right hand side has an almost identical location in all four complexes. In contrast, the catecholate unit shown on the left, in essentially the same position in 5-, 6-and 8-Lic, is pulled upwards by the shorter linker in 4-Lic; nevertheless, its two iron-chelating oxygen atoms remain in positions very close to those seen in the other three structures. The structural differences can be quantified by calculating the mean planes defined by the six carbon atoms of the two catecholate rings, and measuring their inter-planar angle which is 97°, 111°, 110° and 110° in CFe-4-Lic, CFe-5-Lic, CFe-6-Lic and CFe-8-Lic, respectively (Fig. 4). It is evident that Fe-4-Lic cannot bind with the optimum inter-planar angle of ~110° due to its short butylene linker. In contrast, the longer and more flexible linkers in Fe-5-Lic, Fe-6-Lic and Fe-8-Lic can adapt to both the geometric requirements of the Fe(III) center and the binding pocket of CeuE. Crystal structures of the mutants. All five mutants (Y288F, H227L, H227A, H227L/Y288F, H227A/ Y288F) were crystallized in their apo form, and all structures are closely similar to that of wild type CeuE. All mutations were confirmed by sequencing of the DNA. However while there is clear electron density for the Y288F mutation in its three structures, H227L and H227A are located in a flexible loop region and there is generally no electron density to confirm their presence. Co-crystallization of mutant H227L with Fe-5-Lic yielded a structure containing three protein ligand complexes in the asymmetric unit (PDB ID: 5TCY). The loop region upon which the H227L mutation is ordered, but points away from the Fe(III) coordination sphere. A water molecule completes the octahedral Fe(III) coordination (with good density in only one of the three independent molecules in the asymmetric unit, Fig. 5).
Intrinsic fluorescence quenching. The affinities of CeuE for the four Fe-n-Lic complexes were quantified using intrinsic fluorescence quenching, Table 1, with measurements carried out in triplicate. The data were fitted to a 1:1 binding model and analyzed by non-linear regression 26 . Whilst the binding of Fe-5-Lic was too tight to allow the calculation of an accurate dissociation constant, a comparison of the shape of the individual titration curves suggest that Fe-5-Lic binds slightly more tightly than either Fe-4-Lic or Fe-6-Lic ( Fig. 6(a)). An attempt to determine the K d value of Fe-5-Lic by using a more dilute protein solution was unsuccessful because of the inherent decrease in emission intensity reaching the detection limit of the fluorimeter.
The K d values of Fe-4-Lic and Fe-6-Lic are similar and lie within three standard deviations of one another. This indicates that the entropic cost incurred upon binding of the ligand with the more flexible 6-atom linker is compensated by its ability to accommodate the preferred octahedral coordination geometry of Fe(III). Since the inter-planar angle between the catecholamide units in the CFe-6-Lic and CFe-8-Lic structures is identical (110°), the higher K d value obtained for Fe-8-Lic is likely to reflect the higher entropic cost upon binding due to the greater number of conformational degrees of freedom in its linker. Overall, the relative similarity of the dissociation constants for the series shows that the linker length has only a subtle effect on binding affinity to CeuE.
The binding studies with wild type CeuE were followed by titrations to determine the ligand affinity of all five mutants. Fe-5-Lic was chosen because of its strong binding to wild type CeuE and because its linker length matches that of natural catecholate siderophores. Mutation of the Fe(III)-coordinating tyrosine led to a drastic drop in binding affinity in both the single (Y228F) and the double mutants (H227L/Y288F and H227A/Y288F), with K d values above 1 μ M. In contrast, the effect of mutating the histidine was less pronounced. Mutants H227L and H227A gave K d values of 22 ± 10 nM and 35 ± 14 nM, respectively, indicating only slightly weaker binding than for wild type, which indicates that the tyrosinate O-donor binds the Fe(III) cation more strongly than the histidine N-donor. However, there is an additional entropic contribution to consider. Comparison of the apo structure of CeuE (PDB ID: 3ZKW) with that of CFe-5-Lic (PDB ID: 5A5D) shows that upon ligand binding, Tyr288 only has to shift slightly to coordinate, whilst His227, located on a flexible loop, has to move significantly to reach the Fe(III) center (Fig. 7). Hence, the coordination of the latter will have an entropic cost associated with it.
Subsequently, titrations were carried out with Fe-4-Lic, Fe-6-Lic and Fe-8-Lic to identify trends in the affinity for mutants H227L and H227A ( Fig. 6(b) and (c)) ( Table 1). The resulting K d values show trends similar to that observed for wild type, but with systematically weaker binding. To confirm that the preference for the Λ -configuration is retained in solution, circular dichroism (CD) spectroscopy was used to probe the ligand-to-metal charge transfer (LMCT) band ( Fig. 8(a)). The minimum and maximum of the region of the LMCT band at approximately 408 nm and 595 nm, respectively, are indicative of the Λ -configuration 29 , with no CD signal observed in the absence of either the protein or ligand, confirming that the protein selectively binds the Λ -configuration. The decrease in amplitude of the signal with the increase in the number of spacer atoms indicates that CeuE binds the ligands in the order Λ -Fe-5-Lic > Λ -Fe-6-Lic > Λ -Fe-8-Lic, in agreement with the dissociation constants recorded by fluorescence titration.
Circular dichroism spectroscopy was used to investigate if the mutants also induced Λ -configuration in Fe-5-Lic. The Y228F mutants gave no signal, confirming a lack of Λ -configuration induction, which may indicate a lack of ligand binding. The Λ -configuration signal was observed for mutants H227L and H227A, indicating that these do indeed bind Λ -Fe-5-Lic ( Fig. 8(b)). Complexes of H227L and H227A were then prepared with all four Fe-n-Lic complexes. The Λ -configuration was observed for all four, but with a decrease in signal amplitude compared to wild type (Fig. 8(c)), confirming the reduced binding affinity observed in the fluorescence quenching experiments.

Summary and Conclusions
The tetradentate siderophore mimics Fe-5-Lic, Fe-6-Lic and Fe-8-Lic were co-crystallized with the periplasmic binding protein CeuE of C. jejuni. In addition to electrostatic interactions and hydrogen bonding, the interactions of the binding pocket of CeuE with all three siderophore complexes involved the direct co-ordination of two amino acid side chains (His227 and Tyr288) to the Fe(III) center, as previously observed for CFe-4-Lic. The inter-planar angle between the Fe(III)-coordinated catecholamide units increases from a strained 97° found in CFe-4-Lic to 111°, 110° and 110° in CFe-5-Lic, CFe-6-Lic and CFe-6-Lic, respectively. As in our previously determined co-crystal structures, CeuE was found to select for Λ -configured Fe(III) complexes in all these structures. In addition, the affinities of CeuE for Fe-4-Lic, Fe-5-Lic, Fe-6-Lic and Fe-8-Lic were determined using fluorescence quenching, which yielded dissociation constants of 21 ± 6 nM, < 10 nM, 33 ± 8 nM and 58 ± 8 nM, respectively. These results indicate that CeuE exhibits a degree of ligand-binding promiscuity and that the length of the linker between the Fe(III)-binding catecholamide units has a subtle effect on the binding of the bis(catecholate) siderophores, which is driven by the balance between the enthalpic gain through optimized interactions with the binding pocket vs entropic costs associated with the loss of conformational degrees of freedom. Accordingly, the binding affinity initially increases with the increase in linker length from 4 to 5 before it decreases upon extending further to 6 and 8 linker atoms. Not surprisingly, the optimum linker length of five corresponds to that found in natural siderophores that contain lysine or di-serine-based linkers, such as enterobactin and azotochelin.
Site-directed mutagenesis of CeuE revealed that Y288 is essential for binding, as the binding affinity observed in the fluorescence titration experiments is far weaker for all mutants containing Y228F when compared to the wild type protein. This finding is confirmed by the lack of CD signal corresponding to the Λ -configuration when Fe-5-Lic is added to these mutants. Mutant H227L retained the capacity to bind ligands, though with reduced affinity compared to wild type, Table 1, suggesting the histidine is not essential to binding. This is supported by the structure of the H227L mutant in complex with Fe-5-Lic, where a water molecule completes the octahedral coordination of Fe(III). Circular dichroism indicated that the Λ -configured ligand arrangement observed in the crystal structure is maintained in solution phase, and that the Λ -configuration is selected by H227L for all Fe-n-Lic species, but with a weaker signal than for wild type CeuE, due to weaker binding affinities across the board.

Synthesis of 5-LICAM, 6-LICAM and 8-LICAM.
The ligands were prepared as previously reported 30,31 . Cloning, expression and purification. Wild type CeuE. All proteins were expressed by standard procedures previously described 20 . The numbering used for C. jejuni CeuE starts at the first residue of the mature protein after cleavage of the 20 amino acid signal peptide 20 . The construct 24-310 in our numbering corresponds to residues 44-330 from the Uniprot entry Q0P8Q4_CAMJE for the full length precursor. Four residues (GlyProAlaMet) remain at the N terminus of the construct after 3 C cleavage and the naturally occurring residues start at Leu24.
Protein purification involved standard Ni-NTA-agarose column chromatography to purify His-tagged CeuE. The tag was then cleaved with 3 C protease and dialyzed overnight against 20 mM Tris, pH 7.5, 150 mM NaCl. Final purification involved a second 5 mL Ni-NTA-agarose column with protein elution via an imidazole gradient. Non-tagged protein was eluted, concentrated by centrifugal ultrafiltration (Amicon Ultra) and purified by size exclusion chromatography, using a 120 ml Hi load 16/60 Superdex 200, preparative grade column in 20 mM Tris, pH 7.5, 150 mM NaCl. The resulting samples were concentrated to 80 mg mL −1 .
The plasmid DNAs of the CeuE single mutants were then subjected to a second cycle of PCR to yield the double mutants H227L/Y288F and H227A/Y288F. The mutant proteins were expressed and purified according to the standard procedure detailed for wild type CeuE. Pure mutants were concentrated to 20-40 mg/ml and stored at − 80 °C.

Characterization of apo CeuE mutants. Circular dichroism spectroscopy confirmed that all mutants
were correctly folded. Electrospray ionization mass spectrometry was used to confirm that all mutations were correctly incorporated. Experimental MWs were within 1 Da of the expected value.
Crystallization. CFe-n-Lic co-crystals. Protocols analogous to those used in the preparation of CFe-4-Lic were followed 20 . For co-crystallization, a solution of CeuE was diluted to 20 mg mL −1 in a buffer of 20 mM Tris, 150 mM NaCl at pH 7.5. 114 μ L of this solution were mixed with 6 μ L of the Fe-5-Lic stock solution to give a 1:10 molar ratio, and 4 mL of protein buffer were added to ensure all the ligand remained in solution. To wash out the excess ligand, additional buffer was added and the diluted solution was re-concentrated using Amicon Centrifugal filter units to a final protein concentration of 20 mg mL −1 (measured by following the Bradford method). The solution was centrifuged to remove precipitate, and concentrated to a final volume of ~400 μ L. For Fe-6-Lic, 57 μ L of CeuE diluted to 20 mg mL −1 was mixed with 3 μ L of the Fe-6-Lic stock solution to give a 1:10  : 5A5D). Tyr288, located on a helical region, has only to shift slightly to coordinate, whilst His227, located on a flexible loop, has to move significantly to reach the Fe(III) center.
Screening was performed in 96-well plates in a Mosquito nanoliter pipetting robot (TTP LabTech, UK) using sitting-drop vapor diffusion with commercial PACT (Molecular Dimensions) and JCSG screens. Each drop contained 150 nL complex solution and 150 nL reservoir solution. For CFe-5-Lic the best diffraction was observed for crystals grown from two similar PACT conditions, H6 (0.2 M sodium formate, 0.1 M BisTrisPropane, pH 8.5, 20% PEG 3350) and H12 (0.2 M sodium malonate, 0.1 M BisTrisPropane, pH 8.5, 20% PEG 3350). A crystal from condition H12 was used for data collection. For CFe-6-Lic the best diffraction was observed for those grown from two similar PACT conditions, C3 (0.1 M PCB buffer comprising 2:1:2 molar ratio sodium propionate, sodium cacodylate, and BisTrisPropane, pH 6, 25% PEG 1500) and C4 (0.1 M PCB buffer comprising 2:1:2 Structure determination. Data were collected at the Diamond Light Source. Computations were carried out using programs from the CCP4 suite 32 , unless otherwise stated. Diffraction images were processed using XIA2 [33][34][35] . The structures were solved with MOLREP 36 or PHASER 37 , and refined by REFMAC 38 iterated with manual model building/correction in COOT 39 . Validation was performed using PROCHECK 40 and MOLPROBITY 41 . CFe-n-Lic complexes. The CFe-5-Lic crystal was cryo-protected with 35% PEG 3350 and vitrified at 110 K. The CFe-6-Lic and CFe-8-Lic crystals were cryo-protected with 32.5% PEG 1500 and 32.5% PEG 3350 respectively and vitrified at 110 K. H227L-Fe-5-Lic was cryo-protected with 32.5% PEG 1.5 K vitrified at 110 K. The CFe-5-Lic and CFe-6-Lic were isomorphous in space group P2 1 2 1 2 1 , with a single protein monomer in the asymmetric unit. The  CFe-8-Lic crystal was also in space group P2 1 2 1 2 1 , but with different cell dimensions and crystal packing. H227L-Fe-5-Lic was in space group P1. The structures were solved starting with CFe-4-Lic (PDB ID: 5A1J) as a search model. Restraints for the ligands were modelled using JLigand 42 . Crystallographic statistics are detailed in Table 2.
CeuE mutants. All mutant crystals were cryo-protected with well solution containing 32.5% of relevant PEG and vitrified at 110 K. H227L, H227A, H227L/Y288F and H227A/Y288F were in space group P1 with three protein monomers in the asymmetric unit. Y288F was in space group P3 2 21 with one protein monomer in the asymmetric unit. Structures were solved starting with apo wild type CeuE (PDB ID: 3ZKW) as a search model. The overall protein fold for each mutant was essentially identical to that of wild type CeuE. There was no well-defined electron density for a small number of surface loops, indicating these regions were disordered in the crystal. There was clear electron density confirming the presence of phenylalanine in place of tyrosine in all three Y288F containing structures. However, H227 is located on a flexible loop and the electron density for the H227A and H227L side chain was absent in these structures. Crystallographic statistics for all mutant structures are detailed in Tables 3 and 4. Fluorescence spectroscopy. Fluorescence quenching studies with CeuE were carried out on a Hitachi F-4500 fluorescence spectrophotometer as previously reported 22 .
A 240 nM solution of CeuE in 40 mM Tris-HCl pH 7.5, 150 mM NaCl was prepared and the concentration verified by both the Bradford method and UV-vis absorbance at 280 nm, using the molar absorbance coefficient predicted by ProtParam (15930 mol −1 dm 3 cm −1 ) 43 . The CeuE solution (2 mL) was titrated stepwise with 16 aliquots of a 12 μ M Fe-n-Lic solution resulting in a concentration range of Fe-n-Lic of 0 to 1.19 μ M. The observed emission intensity was corrected for photomultiplier tube response and buffer subtracted, then integrated between 310 and 410 nm. The data were normalized and analyzed by nonlinear regression using a one-site binding model in DynaFit 26 . Each system was analyzed in triplicate and the average dissociation constants were calculated as reported previously 44 .   The spectra were recorded from 300-700 nm and buffer subtracted (data pitch 0.5 mm, scan speed 100 nm min −1 , response 2 seconds, bandwidth 2 nm, path length 1 cm). The spectra were recorded 5 times and averaged.


extfieldsofstudy: Medicine Chemistry

text:
A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TES148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TES148C and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition–elimination (TEWT) to a lower energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.

Structure Determination
Colorless needles of S2 were grown from a hexanes solution of the compound at -30 deg. C. A crystal of dimensions 0.26 x 0.01 x 0.01 mm was mounted on a Rigaku AFC10K Saturn 944+ CCD-based X-ray diffractometer equipped with a low temperature device and Micromax-007HF Cu-target micro-focus rotating anode (λ = 1.54187 A) operated at 1.2 kW power (40 kV, 30 mA). The X-ray intensities were measured at 85(1) K with the detector placed at a distance 42.00 mm from the crystal. A total of 2633 images were collected with an oscillation width of 1.0° in ω. The exposure times were 15 sec. for the low angle images, 75 sec. for high angle. The integration of the data yielded a total of 98528 reflections to a maximum 2θ value of 136.48° of which 9383 were independent and 8176 were greater than 2σ(I). The final cell constants (Table  1) were based on the xyz centroids 44319 reflections above 10σ(I). Analysis of the data showed negligible decay during data collection; the data were processed with CrystalClear 2.0 and corrected for absorption. The structure was solved and refined with the Bruker SHELXTL (version 2008/4) software package, using the space group P2(1)2(1)2(1) with Z = 4 for the formula C25H35NO7.
There are two crystallographically independent molecules per asymmetric unit. All non-hydrogen atoms were refined anisotropically with the hydrogen atoms placed in idealized positions. Full matrix least-squares refinement based on F2 converged at R1 = 0.0518 and wR2 = 0.1275 [based on I > 2sigma(I)], R1 = 0.0591 and wR2 = 0.1328 for all data. Additional details are presented in Table 1

Kinetic analysis of thioesterase catalysis
Product formation for all kinetic experiments was quantified by linear regression using equations derived from fitting the peak areas of the corresponding standard curves. Standard curves were generated by analyzing product standards in triplicate at a range of concentrations and were linear in all cases. Prior to performing kinetic analysis, the pH dependence for TE macrolactonization was established by running a series of assays at pH 6, 6.5, 7, 7.2, 7.5, 7.8, 8. The reactions consisted of sodium phosphate buffer (50 mM), 1 μM Pik TE (WT or S148C), 1 mM 3 and 10% (v/v) DMSO in a total volume of 50 μL. The reactions were incubated at RT for 1 h and quenched by addition of MeOH (150 μL). All reactions were run in duplicate and following centrifugal clarification (17,000 x g, 15 min, 4˚C) the samples were directly analyzed for product formation, pH 7.2 was found to be optimum.

Macrolactonization of 4:
Macrolactonization of SNAC-MeHK 4 to 6 was monitored via analytical high performance liquid chromatography (HPLC) using an Agilent 1100 Series HPLC and a Zorbax SB-Phenyl (3.5 µm, 4.6 x 150 mm, part number 863953-912) column at a wavelength of 250 nm. Products were separated using a linear gradient via the following method: 3.0 mL/min, solvent A: H 2 O, solvent B: MeCN, 20% B 0-1 min, 20-60% B linear gradient 1-10 min, 100% B 10-11 min, 20% B 11-12 min. A time course for the macrolactonization reaction of each TE at pH 7.2 was obtained by analyzing reaction mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.2 mM 4, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 1500 μL incubated at room temperature for intervals of 5, 10, 20, and 30 min at which point they were quenched and analyzed via HPLC for macrolactone production. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 20 min ( Figure S6).
Steady-state kinetic analysis was performed by determining the initial velocities for the macrolactonization of 4 at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM. The reactions consisted of 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, variable concentrations of 4, and 0.3 μM TE S148C or 1.0 μM TE WT . The reactions were incubated at room temperature for 10 min at which point they were quenched with MeOH (150 μL) and analyzed via HPLC for macrolactone production. All reactions were run in triplicate and the initial velocities at different substrate concentrations were fit to the Michaelis-Menten equation by nonlinear least-squares regression to calculate k cat and K M ( Figure S7). The 6 mM and 8 mM concentration points were omitted from the plots with TE S148C as these concentrations resulted in significant substrate inhibition.

Hydrolysis and macrolactonization of 5:
The hydrolysis (TE WT ) and macrolactonization (TE S148C ) of 5 to either 7 or 9, respectively, was monitored via HPLC using the same method as above for 4. A time course for the hydrolysis and cyclization reaction for each TE was accomplished by incubating assay mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.03 mM (TE S148C ) or 0.125 mM (TE WT ) 5, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 1500 μL at room temperature for intervals of 5, 10, 20, and 30 min, at which point they were quenched and analyzed via HPLC. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 30 min ( Figure S8).
Steady-state kinetic analysis was performed by determining the initial velocities for the macrolactonization and hydrolysis of 5 at concentrations of 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM for TE WT and 0.03 mM, 0.06 mM, 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2mM, 3 mM, 4mM, 6mM, 8mM for TE S148C . The reactions consisted of 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, variable concentrations of 5, and 0.5 μM TE S148C or 1.0 μM TE WT . The reactions were incubated at room temperature for 10 min at which point they were quenched and analyzed via HPLC for product formation. All reactions were run in triplicate and the Initial velocities at different substrate concentrations were fit to the Michaelis-Menten Figure S7. Steady state kinetic analysis for the TE catalyzed macrocyclization of 4. Figure S8. Linear velocity determination for the hydrolysis (WT) and macrocylization (S148C) of 5. S13 equation by nonlinear least-squares regression to calculate k cat and K M ( Figure S9). The concentration points not represented were removed from the plot as they resulted in significant substrate inhibition.

Macrolactonization of 3:
Macrolactonization of the native hexaketide 3 was monitored via analytical high performance liquid chromatography (HPLC) using an Agilent 1100 Series HPLC and a Zorbax SB-Phenyl (3.5 µm, 4.6 x 150 mm, part number 863953-912) column at a wavelength of 250 nm. Products were separated using a linear gradient via the following method: 3.0 mL/min, solvent A: H2O, solvent B: MeCN, 10% B 0-1 min, 10-40% B linear gradient 1-10 min, 100% B 10-11 min, 10% B 11-12 min. A time course for the cyclization reaction with each TE was accomplished by incubating assay mixtures containing 400 mM phosphate buffer (pH 7.2), 10% (v/v) DMSO, 0.125 mM 3, and 0.3 μM TE S148C or 1.0 μM TE WT in a total reaction volume of 350 μL at room temperature for intervals of 5, 10, 20, and 30 min, at which point they were quenched and analyzed via HPLC. The NBOM protected substrate was deprotected and utilized in enzymatic reactions as previously reported 1 , briefly: a solution of ascorbic acid (25 mM final concentration), sodium metabisulfite (1 mM final concentration), NBOM protected hexaketide (1 mM final concentration), and H 2 O (requisite dead volume) was irradiated under a consumer facial tanning lamp at a height of 14 cm (Verseo #AH129c) for 20 min to furnish the deprotected Pik hexaketide 3. After photolysis, the solution was diluted with enzymatic reaction buffer, and catalysis was initiated via the addition of enzyme. Reactions were run in duplicate and the velocities were determined to be linear by linear regression analysis of the data over a period of 20 min ( Figure S10).
Samples were quantified by linear regression analysis comparing the product peak areas to those generated from the corresponding standard curves.

Pik TE structure preparation
The wild type apo Pik TE structure was obtained from the RCSB Protein Data Bank (PDB: 1MNA) 9 . Structures for the wild type and mutant enzymes were prepared from WT Pik TE using PyMOL 1.3 10 . Chain B of PDB file 1MNA and the crystallographic water were removed, and 5 missing protein residues (T109-T113) were built into chain A using PyMOL 1.3. 10 Parameters for the apo TE were generated using the antechamber module of AMBER 14 11 with the widely tested Stony Brook modification of the Amber 99 force field (ff99SB) 12 force field. The initial substrate conformations were generated by modeling the linear hexaketide chain as neutral acyl serine or cysteine amino acids with N-terminal (-CO-CH 3 ) and C-terminal (-NH-CH 3 ) caps in an approximately cyclic orientation using the solved crystal structures of the derivatized macrolactone products as templates. The structure reported in reference 1 served as the template for Pik TE-4 and S2 was used for modeling the Pik TE-5. The initial hexaketide conformations were then optimized by performing a geometry optimization with the M06-2X functional 13 level using the Gaussian 09 package 14 . The charges and parameters for the hexaketide substrates were then determined using the general AMBER force field (GAFF) 15 with partial charges set to fit the electrostatic potential generated at the HF/6-31G(d) level by the RESP model 16 . The charges were calculated according to the Merz-Singh-Kollman scheme 17,18 , using Gaussian 09. The N and C-terminal caps were then removed and the parameterized hexaketides were used to replace Ser148 of the TE to generate Pik TE modeled as acyl-enzyme intermediates. Parameter, topology, and coordinate files for the complex were compiled for molecular dynamics (MD) simulation using the tleap 11 module. Following parameterization, the Pik TE models with bound hexaketides were immersed in a preequilibrated truncated cuboid box with a 10 Å buffer of TIP3P 19 water molecules using tleap and the systems were neutralized by addition of explicit counter ions (Na + and Clˉ).

Molecular Dynamics simulations
MD simulations were performed using the AMBER 14 package. 11 A cutoff of 8 Å was used for all Lennard-Jones and electrostatic interactions. Water molecules were treated with the SHAKE algorithm such that the angle between the hydrogen atoms was kept fixed. Periodic boundary conditions simulated the effects of a larger system size. Long-range electrostatic effects were modelled using the particle-mesh-Ewald method. 20 Prior to performing the production MD simulations, the solvated systems were subjected to two steps each of minimization and equilibration to relax and correct any possibly unrealistic arrangements: two minimizations were performed first over a total of 5000 cycles each, with the method being switched from the steepest descent algorithm to conjugate gradient after 2500 cycles. The first minimization relaxed the positions of the solvent molecules and ions by imposing positional restraints on the solute by a harmonic potential with a force constant of 500 kcal mol -1 Å -2 . Following the first minimization, a distance restraint of 2.3 Å between the hexaketide nucleophilic hydroxyl hydrogen and the Nε nitrogen of His268 was imposed and maintained throughout the second minimization, both equilibrations, and first production simulation, the restraint was then released S20 for the second production simulation. This restraint maintains the hexaketide and the catalytic histidine residue -His268 acts as the general base-in an arrangement close to that calculated for the macrolactonization transition state, while allowing the hexaketide backbone to explore both reactive and non-reactive conformations. The restraint also served to minimize any potential biases resulting from the initial substrate positioning. After the distance restraint was imposed, a second stage minimization was performed on all the atoms in the simulation cell without cartesian atomic restraints. Post minimization, two equilibrations were performed. In the first equilibration step, a 0.1 ns MD simulation was performed with a constant pressure of 1 atm and a constraint of 10 kcal mol -1 Å -2 on the protein and substrate. The Andersen thermostat equilibration scheme was used to control and equalize the temperature by gradually increasing the system temperature from 0 to 300 K over the course of the first equilibration run. The system volume was then fixed and a second equilibration step was achieved with 2 ns MD simulation conducted without cartesian atomic restraints with a temperature maintained at 300 K using the Andersen thermostat.
Post equilibration, two additional MD simulations were carried out utilizing the above conditions: Initially, a 50 ns simulation was performed with the aforementioned distance restraint intact to ensure proper shape complementarity of the TE active site for the acyl intermediate forced in a pre-catalytic state. Next, the substrate-protein distance restraint was removed and a final production simulation was run for an additional 500 ns. This portion of the simulation represents the conformation of both the substrate and the protein environment in the covalently bound Michaelis-Menten complex prior to reaction. An integration time step of 2 fs was utilized with structural snapshots being extracted every 50 steps during both production simulations. The resulting production trajectories were analyzed using the cpptraj program in Ambertools14 employing the default options to describe hydrogen-bonding, hydrophobic interactions, and clustering analysis. Clustering analysis of the resulting simulations based on the conformation of the acyl-enzyme intermediate was used to bin each frame into representative clusters of structures. The clusters were subsequently examined for hydrogen-bond and hydrophobic interactions involving the substrate and TE using LigPlot+ 21 ( Figure S13).
Examination of the overall protein root-mean-square deviation (RMSD) and per-residue rootmean-square fluctuation (RMSF) values for the trajectories of each TE complex demonstrated stable dynamic behavior throughout the course of the simulations, with Pik TE-5 maintaining a slightly increased level of flexibility ( Figure S14). Investigation of the Pik TE-5 residues that displayed higher RMSF values revealed these residues were located predominantly in loops and linker regions with higher intrinsic flexibility and had limited impact on the conformation of the hexaketide. Pik TE-4 clusters I and II stabilize the hexaketide in a catalytically productive conformation mainly by hydrophobic packing leading to a high level of shape-complementarity and hydrogen bonding between the nucleophilic hydroxyl and the catalytic histidine. Conversely, cluster III shows decreased hydrophobic contacts and the hexaketide adopts a more linear conformation making it unreactive towards macrolactonization. Pik TE-5 Cluster I' stabilizes the hexaketide in a cyclic conformation via hydrophobic packing; however the hexaketide adopts a conformation that impedes macrocyclization due to formation of an intramolecular hydrogen bond. Clusters II' and III' show a gradual loss of hydrophobic packing as the hexaketide adopts a linear conformation that makes it vulnerable to the experimentally observed hydrolysis. The catalytic triad His268 and Asp176 residues are colored yellow while the active site residues found to interact with the acyl-intermediate are colored green. Residues involved in hydrophobic interactions are displayed in surface representation; dashed lines indicated hydrogen bonds to the labeled residues.

QM Calculations
TE catalyzed macrolactonization QM Computational Details. Full geometry optimizations were carried out with Gaussian 09 14 using the M06-2X hybrid functional 13 and 6-31+G(d,p) basis set. Solvation was considered implicitly through the PCM polarizable continuum model 22 in order to better reproduce the electrostatic environment inside the enzyme active site (ε=4). The possibility of different conformations was taken into account for all structures. All stationary points were characterized by a frequency analysis performed at the same level used in the geometry optimizations from which thermal corrections were obtained at 298.15 K. Local minima and first order saddle points were identified by the number of imaginary vibrational frequencies. The quasiharmonic approximation reported by Truhlar et al. was used to replace the harmonic oscillator approximation for the calculation of the vibrational contribution to enthalpy and entropy. 23 Scaled frequencies were not considered. Mass-weighted intrinsic reaction coordinate (IRC) calculations were carried out by using the Gonzalez and Schlegel scheme 24,25 in order to ensure that the TSs indeed connected the appropriate reactants and products. Gibbs free energies (ΔG) were used for the discussion on the relative stabilities of the considered structures. Cartesian coordinates, electronic energies, entropies, enthalpies, Gibbs free energies, and lowest frequencies of the calculated structures are available below.
Attempts to locate transition states for the initial covalent binding step (i.e. Ser/Cys148 acylation) from thioester models of Pik TE WT and Pik TE S148C were unsuccessful, due to the very flat topology found for the corresponding potential energy surfaces. Hence, very low activation barriers are expected for the initial substrate binding, and macrocyclization was assumed to be the rate-limiting reaction.


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Proteomic Applications of Polymeric Nanoparticles with Engineered Affinity towards Select Target

Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by a variety of methods. Selective precipitation is perhaps the simplest method for separating one type of macromolecule from another. Most purification methods, however, involve some form of chromatography whereby molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase). Gel filtration (also called size-exclusion chromatography or SEC) uses a porous resin material to separate molecules based on size (i.e., physical exclusion). In ion exchange chromatography, molecules are separated according to the strength of their overall ionic interaction with a solid phase material (i.e., nonspecific interactions).

Introduction to Affinity Purification
Proteins and other macromolecules of interest can be purified from crude extracts or other complex mixtures by a variety of methods. Selective precipitation is perhaps the simplest method for separating one type of macromolecule from another. Most purification methods, however, involve some form of chromatography whereby molecules in solution (mobile phase) are separated based on differences in chemical or physical interaction with a stationary material (solid phase). Gel filtration (also called size-exclusion chromatography or SEC) uses a porous resin material to separate molecules based on size (i.e., physical exclusion). In ion exchange chromatography, molecules are separated according to the strength of their overall ionic interaction with a solid phase material (i.e., nonspecific interactions).
By contrast, affinity chromatography (also called affinity purification) makes use of specific binding interactions between molecules. It is a variant of chromatography based on the ability of biomolecules (analytes) to bind certain ligands specifically and reversibly. These unique features of the analyte and the ligand interaction are then utilized for the separation of the analyte of interest from a complex mixture. From the first protein-protein interaction studies done in the late 1990´s [1,2], affinity separations have experienced a true renaissance in proteomics. A complete parade of affinity matrices and affinitybased experimental approaches has been developed that has found numerous applications ranging from subtraction of highly abundant proteins to study of drug target profiles to large scale mapping of posttranslational modifications. In the classical setup, a relevant ligand is attached to a solid, inert resin creating an affinity stationary phase (affinity matrix). When a sample containing desired analyte is passed over such affinity matrix, the analyte having specific binding affinity to the ligand become bound and retained by the matrix while the other molecules stay apart. After the other unwanted molecules are washed away, the bound analyte is stripped from the affinity matrix, resulting in its purification from the original sample ( Figure 1). This principle was discovered by Cuatrecasas and Wilchek [3,4], who applied it to the purification of Staphylococcal nuclease and avidine. Since their discovery, numerous specialized affinity purification techniques appeared, but notably even today more than 90% of them apply the same general principles as reported in 1968 [5]. Each specific affinity system requires its own set of conditions and presents its own peculiar challenges for a given research purpose [6]. Volume 5 Issue 4 -2017 Basically, there are two main modes of affinity chromatography -a 'subtraction' mode and an 'enrichment' mode. If the aim of the purification is to specifically remove protein species that would hamper characterization of the sample, then affinity chromatography is employed in subtraction mode. Traditionally, the main purpose of affinity subtraction is the elimination of highly abundant proteins like albumin, immunoglobulin, etc., to achieve broader coverage of proteomes that suffer from wide dynamic range such as human body fluids (blood plasma, cerebrospinal fluid, urine, saliva). Whereas, enrichment mode is applied for the isolation of selected protein species. At the protein level, the enrichment affinity chromatography permits the purification of a particular protein of interest or a group of low abundant proteins and/or proteins that share a specific structural feature. At peptide level, the enrichment affinity chromatography has attained an essential position in the purification of post-translationally modified species.
Moreover, the enrichment affinity chromatography of proteins can be used as a tool to obtain an information on specific protein affinities. In short, the basic principle of affinity chromatography allows to utilize the method for the isolation of protein partners of selected molecules [7,8]. During the affinity purification, the molecule of interest represents a 'bait' that is bound by its cellular protein counterparts, its 'preys'. These (protein) preys are then easily purified and consequently identified by MS [9,10], generating thus a map of the bait-protein interaction network. In this setup, affinity chromatography has facilitated the discernment of many different molecular relationships from protein-protein interactions to drug selectivity profiles [11][12][13].

Engineered polymeric nanoparticles for affinity purification
The support or matrix in affinity purification is any material to which a bio-specific ligand is covalently attached. Typically, the material to be used as an affinity matrix is insoluble in the system in which the target molecule is found. Usually, but not always, the insoluble matrix is a solid. Hundreds of substances have been described and utilized as affinity matrices, including agarose, cellulose, dextran, polyacrylamide, latex and controlled pore glass [14][15][16]. Useful affinity supports are those with a high surface-area to volume ratio, chemical groups that are easily modified for covalent attachment of ligands, minimal nonspecific binding properties, good flow characteristics and mechanical and chemical stability.

PNIPAm-based nanoparticles for GPER purification
G-protein coupled Estrogen Receptor may play a significant role in tamoxifen resistance in breast cancer cells. Although it is believed to be a key player in several other cancers, the background information at the molecular level is still limited. In order to study the complete proteome, post translational modifications, mutations and to better understand their molecular interactions with various other receptors and ligands, it is necessary to isolate pure GPER from crude cell lysate. The lack of promising separation and purification tools peaked our interest towards designing NPs that can capture this target biomolecule. In recent years, NPs with an intrinsic affinity have shown to be successful in binding biomacromolecules like melittin [37,38], immunoglobulin G [39], histone [40], fibrinogen [40] and lysozyme [29] by controlling and optimizing the functional monomers composition.
In our study, we adopted a similar approach in an effort to capture GPER with high affinity and selectivity among a mixture of proteins that are expressed in various breast cancer cell lines. Considering the fact that GPER is a membrane protein with many hydrophobic amino acid residues on the surface, a candidate NP was chosen from a library of nanoparticles that were prepared by combining different populations of functional groups on a poly-N-isopropylacrylamide (PNIPAm)-based polymer backbone. The synthesis of the candidate NPs was illustrated below ( Figure  3). We started our experiment with GPER peptide selection and synthesis. Then, we evaluated the interaction between truncated-GPER (short peptide epitopes) and NPs by high performance liquid chromatography (HPLC). Finally, we moved on to evaluate the interaction between GPER (whole protein) and NPs by immunoblot analysis. After evaluating the results, the nanoparticle-based purification approach turned out to be the method of choice to enrich GPER prior to mass spectrometry analysis.

Conclusion
Recently, nanoparticles with an intrinsic affinity have shown to be successful in binding biomacromolecules like melittin, immunoglobulin G, histone, fibrinogen and lysozyme by controlling and optimizing the functional monomers composition. We tested a similar approach in an effort to capture GPER with high affinity and selectivity among a mixture of proteins that are expressed in various cancer cell lines. We anticipate to use this strategy as a sample purification step before mass spectrometrybased proteomic analysis. Similar approach could be applied to study a number complex targets that are difficult to purify or lack separation tools with current proteomic technologies.