s2fieldsofstudy: Biology Chemistry

text:
Interaction of Aryl Hydrocarbon Receptor-interacting Protein-like 1 with the Farnesyl Moiety*

Background: Mutations in AIPL1, a chaperone of the lipidated visual effector phosphodiesterase-6, cause severe childhood blindness. Results: AIPL1 binds the farnesyl lipid moiety. The unique insert region of AIPL1 is critical for this interaction. Conclusion: The AIPL1-farnesyl interaction suggests its role in the interaction with phosphodiesterase-6 and normal function of AIPL1. Significance: This study describes a novel mechanism of AIPL1 in retina disease. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

Leber congenital amaurosis (LCA) 3 is a severe, early onset, inherited retinopathy that accounts for ϳ5% of all inherited retinopathies and is one of the main causes of blindness in children (1,2). To date, 14 genes have been identified as being mutated in LCA patients, including the gene Aipl1 (aryl hydrocarbon receptor-interacting protein-like 1) (3,4). Aipl1 mutations result in one of the most clinically severe forms of LCA (LCA type 4).
AIPL1 is expressed selectively in the retina and the pineal gland (3,5). AIPL1 shares high sequence homology (49% identity) and similar domain organization with the ubiquitously expressed aryl hydrocarbon receptor-interacting protein (AIP) (3). AIP and AIPL1 contain an N-terminal FK506-binding protein (FKBP)-like domain and a C-terminal tetratricopeptide repeat (TPR) domain with three tetratricopeptide repeats (3, 6) (Fig. 1A). These domains are often found in proteins with chaperone activity (7). In addition, in primates, AIPL1 proteins carry at the C termini a proline-rich region of poorly understood function. LCA-linked AIPL1 mutations are found in all three domains: the FKBP-like, TPR, and the proline-rich regions (Fig.  1A). These mutations include missense, nonsense, and deletion mutations (8 -10). AIP is a co-chaperone with Hsp90 in the maturation of the aryl hydrocarbon receptor (11), similar to the co-chaperone function of the FKBP and TPR domain-containing immunophilins FKBP51/52 that act on steroid hormone receptors (12). Furthermore, AIPL1 itself was found to interact with Hsp90 and Hsp70 (13). Thus, the function of AIPL1 as an essential component of a retina-specific chaperone complex has been widely hypothesized (3,7,14,15). AIPL1 has been shown to interact with Nedd8 ultimate buster protein 1 (NUB1), implicated in cell cycle regulation and protein degradation (16). However, studies of AIPL1-deficient mouse models provided compelling evidence for phosphodiesterase 6 (PDE6) as the critical photoreceptor partner of AIPL1 (17,18). AIPL1 knock-out mice exhibited rapid severe degeneration of photoreceptors and had no measurable electroretinograms (17). This phenotype is consistent with the manifestation of LCA4 in humans (10,17). Biochemical analysis revealed that these mice had markedly reduced levels and activity of PDE6 prior to degeneration (17). The drastic reduction in PDE6 was specific, as the levels of other main photoreceptor proteins remained unaffected (17). PDE6 is a central effector enzyme in the pho-* This work was supported, in whole or in part, by National Institutes of Health Grants EY-10843 and EY-12682 (to N. O. A.). 1  totransduction cascade (19,20). Rod PDE6 is a heterotetramer composed of two homologous catalytic subunits (PDE6AB) and two copies of an inhibitory (P␥) subunit (20). Light stimulates phototransduction by activating PDE6AB to hydrolyze a second messenger cGMP and close cGMP-gated channels in the photoreceptor plasma membrane (19,20). Mutations in PDE6AB are known to cause retinal degeneration in humans and animal models by elevating intracellular cGMP concentration and triggering photoreceptor cell death (21)(22)(23)(24)(25). Thus, defective PDE6 function due to mutations in AIPL1 readily explains LCA4. However, the mechanism of AIPL1 action remains poorly understood. Evidence suggests that AIPL1 acts on rod PDE6 posttranslationally and stabilizes the enzyme by preventing its misassembly that leads to rapid protein degradation (26). Proper assembly of PDE6 requires correct folding and prenylation of PDE6A and PDE6B, formation of the catalytic heterodimer PDE6AB, and its association with the P␥-subunits. PDE6A and PDE6B are farnesylated and geranylgeranylated, respectively (27). AIPL1 appears to bind to the PDE6A subunit (26), and the farnesyl modification may be involved in this interaction (28). A yeast two-hybrid screen indicated that AIPL1 interacts specifically with farnesylated retinal proteins (28). Yet, other farnesylated proteins, except PDE6, were unaffected in mouse retina lacking AIPL1 (17). In this study, we investigated whether AIPL1 can directly bind the farnesyl moiety, and we examined the possibility that LCA-linked mutations in AIPL1 may disrupt this interaction. To aid our studies, we analyzed the structure of AIPL1 in solution by SAXS and generated a molecular model of AIPL1 most consistent with the scattering data. Our analysis demonstrated high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thereby revealing a novel function of this domain. Fur-thermore, an LCA4-causing mutation C89R was found to abrogate the probe binding, suggesting an essential role for the farnesyl-mediated PDE6A interaction in normal function of AIPL1.

EXPERIMENTAL PROCEDURES
Materials-AMCA, SE was purchased from Invitrogen. S-Farnesyl-L-cysteine methyl ester was purchased from Enzo Life Sciences. All other chemicals were acquired from Sigma.
Cloning, Expression, and Purification of AIPL1 and Its Domains and Mutants-DNA sequences coding the full-length AIPL1, the N-terminal domain (residues 1-161), or the C-terminal domain (residues 162-328) were PCR-amplified from mouse retinal cDNA and cloned into the pET15b vector using NdeI/BamHI sites. Mutations were introduced using standard QuikChange site-directed mutagenesis protocol. The AIPL1⌬96 -143 mutant was generated by replacing the residues 96 -143 of AIPL1WT with two glycine residues in a twostep PCR procedure. In the first step, the AIPL1(1-95)Gly-Gly(144 -155) sequence was amplified with a forward primer containing the start codon and an NdeI site and a reverse primer corresponding to AIPL1(84 -95)Gly-Gly(144 -155). This PCR product was used as a forward primer in the second PCR amplification with a primer containing the stop codon and a BamHI site. This resulting PCR product was then cloned into the pET15b vector using NdeI/BamHI sites.
The His 6 -tagged AIPL1 proteins were expressed in BL21codon plus Escherichia coli cells by induction with 100 M isopropyl 1-thio-␤-D-galactopyranoside at 16°C overnight. Cell pellets were sonicated on ice (five 30-s pulses) in 50 mM Tris-HCl buffer (pH 8) containing 50 mM NaCl, 50 mM L-arginine, 50 mM L-glutamic acid, 10% glycerol, 15 mM DTT (buffer A), and protease inhibitor mixture (Roche Applied Science). The His 6tagged AIPL1 proteins were purified over Ni-NTA resin (Novagen) using buffer A containing 200 mM imidazole for elution. Ni-NTA affinity-purified AIPL1 proteins were used in fluorescence binding assays directly or after additional purification by ion exchange chromatography on a Mono Q5 column (Bio-Rad). For analysis of the structure of AIPL1 in solution by SAXS, the His 6 -AIPL1 protein was further purified by gel filtration chromatography on a Sephacryl 200 column.
Dynamic Light Scattering-Dynamic light scattering was used to examine the degree of monodispersity of AIPL1 samples. Purified AIPL1 was dialyzed against 50 mM Tris-HCl (pH 8.0) buffer containing 50 mM NaCl, 50 mM L-arginine, 50 mM L-glutamic acid, and 10 mM DTT (buffer B). Analyses of monodispersity of dialyzed AIPL1 samples (4 mg/ml) were performed at 22°C using a DynaPro Nanostar instrument (Wyatt). The data were analyzed using the Dynamics 7.1.7 software.
Homology Modeling-A homology model of the N-terminal FKBP-type domain of AIPL1 (residues 1-157) was generated with Modeler 9.11 (29) using the solution NMR structure of AIP FKBP-like domain as the template (PDB ID code 2LKN) (30). The C-terminal TPR domain of AIPL1 (residues 171-328) was modeled with the hm_build macro in Yasara 12.11.25 (31) that used a number of high resolution TPR domain structures in the PDB to generate an optimized model. These domain models were then superimposed on the respective FKBP and TPR domains from the FKBP52 crystal structure (PDB ID code 1P5Q) (32) and energy-minimized using the em_run macro in Yasara to remove any steric clashes between the two domains. The resulting model with missing domain linker and the N-terminal tag was completed and refined against the SAXS data to generate the molecular model of AIPL1 as described below.
Small Angle X-ray Scattering-SAXS analysis was performed to determine the low resolution solution structure of AIPL1. SAXS data on the His 6 -tagged AIPL1 protein were collected at the Advanced Light Source (12.3.1 SIBYLS beamline) at the Lawrence Berkeley National Laboratories. Three different concentrations of AIPL1 (2.4, 1.6, and 0.8 mg/ml in buffer B) were exposed to the synchrotron radiation for 0.5, 1, 2, and 4 s each. Scattering from the buffer (matched against the samples by overnight dialysis) was subtracted and the sample scattering data analyzed by Primus/QT (33). AutoR g and AutoD max were used to calculate the R g (radius of gyration) and D max (maximum particle dimension from a pairwise distribution function) in Primus/QT, and ab inito envelopes were generated using the GASBOR (34), DAMMIF (35), and DAMMIN (36) programs in the ATSAS 2.4.2-1 package (37)(38)(39). The AIPL1 homology model described above was used in the program BUNCH (40) from the ATSAS package to perform combined rigid-body/ab initio modeling against the SAXS data. The two domains were treated as rigid bodies whereas the connecting linker (residues 158 -170) and the N-terminal 22 residue tag/linker were modeled as flexible dummy residues that together best fit the SAXS data. During fitting, the two domains could move independently of each other restrained by the length of the interdomain linker. The ab initio envelopes generated separately were aligned to the best-fit BUNCH model using supcomb13 (41) and visualized in PyMOL 1.5.0.4 (The PyMOL Molecular Graphics System, Schrödinger, LLC).
Preparation of the Fluorescence Probe Farnesyl-Cys-AMCA-Conjugation of AMCA-X to cysteine farnesyl methyl ester was performed at 25°C for 1 h using, respectively, 6 mM and 2 mM solutions of the reagents in 25% acetonitrile, 25% dimethylformamide, and 100 mM HEPES (pH 7.3). The product, farnesyl-Cys-AMCA, was purified by reverse-phase HPLC. Cys-AMCA was prepared by reacting 1 mM AMCA-X with 3 mM cysteine in 50 mM HEPES (pH 7.5), 50% acetonitrile for 30 min at 25°C.
Fluorescence Assay-Fluorescence assays were performed on a F-2500 Fluorescence Spectrophotometer (Hitachi) in 1 ml of 100 mM HEPES (pH 7.3) or 100 mM PBS (pH 7.5). Fluorescence resonance energy transfer (FRET) assay of farnesyl-Cys-AMCA binding to AIPL1 or mutants was performed with excitation at 280 nm and emission at 440 nM. Farnesyl-Cys-AMCA (150 -250 nM) served as an acceptor of AIPL1 Trp fluorescence. The data were fit with equation for binding with ligand depletion, where F 0 is a basal fluorescence of farnesyl-Cys-AMCA, F is the fluorescence after additions of binding protein, F max is the maximal fluorescence change, B m is a concentration of farnesyl-Cys-AMCA, and X is a concentration of added protein. Concentrations of farnesyl-Cys-AMCA and AIPL1 were determined using ⑀ 350 ϭ 19,000 and ⑀ 280 ϭ 55,000 M Ϫ1 cm Ϫ1 , respectively. Fitting of the experimental data was performed with nonlinear least squares criteria using Prism software (GraphPad). The K d values are expressed as mean Ϯ S.E. for at least three independent measurements.
Circular Dichroism (CD) Analysis-CD analysis was performed to verify the folding and secondary structure of AIPL1 mutants. AIPL1 or mutants were dialyzed against 5 mM sodium phosphate buffer (pH 7.9) containing 0.1 mM DTT. CD spectra were collected on 7 M AIPL1 protein samples in a 1-mm cuvette at 25°C over the range of 300 -180 nm with an interval of 1 nm and scan speed of 100 nm/min using a Jasco J-815 CD spectrophotometer in the Biochemistry Department (University of Iowa) or at the Keck Biophysics Facility (Northwestern University).

RESULTS
Analysis of AIPL1 Structure by SAXS-To guide our analysis of the interaction of AIPL1 with the farnesyl moiety, we examined the structure of mouse AIPL1 in solution by SAXS and generated a molecular model of the protein that agrees the best with the scattering data. With samples that are monodisperse and homogeneous, SAXS data can be used to determine threedimensional envelopes and model the protein using existing high resolution structures of similar proteins in the PDB database (34,42,43). The three-step purification procedure (Niaffinity, ion exchange, and gel filtration) yielded AIPL1 preparation of Ͼ95% purity (Fig. 1, B and C) and excellent monodispersity as measured by dynamic light-scattering (polydispersity 15%). All SAXS scattering curves superimposed quite well, reflecting no concentration dependence. However, scat-tering curves for the high exposures at the highest protein concentration were excluded from analysis due to observable radiation damage. An R g value of 28.91 Å was calculated from the Guinier region (q⅐R g Ͻ 1.3) of the scattering curve ( Fig. 2A) (42). A bell-shaped curve in the low q region of the Kratky plot increasing somewhat in the high q region indicated that AIPL1 is mostly globular with minor presence of unfolded structure (Fig. 2B). Likewise, a plateau at low q in the Debye-Porod plot suggested that the AIPL1 sample has at least a folded core (Fig.  2C). A D max value of 90.81 Å was obtained from the pairwise distribution function, the shape of which also indicated a multidomain protein (Fig. 2D).
Model of AIPL1 Consistent with SAXS Analysis-To reduce modeling bias for AIPL1 from full-length templates (such as FKBP51, PDB ID code 1KT1) (44), we generated homology models for the relatively rigid FKBP and TPR domains individually without the N-terminal and domain connecting flexible linkers. We then used a rigid body fit of these domains against the SAXS data along with ab inito modeling of the flexible linkers to guide us to the relative orientation of these fragments that best explained the experimental x-ray scattering data. This led to a remarkably good model for AIPL1 that fit the SAXS data with a value of 1.17 ( Fig. 2A) and aligns well with the ab initio envelopes generated from the SAXS data alone (Fig. 3A). An overlay of the SAXS-based AIPL1 model with FKBP51 (PDB ID code 1KT1) (44) and FKBP52 (PDB ID code 1P5Q) (32) crystal structures using the FKBP domains for superposition highlights the heterogeneity in relative orientation between the FKBP and TPR domains possible in this class of proteins (Fig. 3B).
The FKBP-like Domain of AIPL1 Directly Binds Farnesylated-Cys Probe-Following S-farnesylation at a protein C-terminal "CAAX box," the three terminal residues are cleaved, and the extreme Cys residue is carboxymethylated (27,45,46). Consequently, S-farnesyl-L-cysteine methyl ester modified at the amino group with AMCA-X represents an appropriate mimetic of the processed C-terminal Cys of farnesylated proteins. A, representative buffer-subtracted scattering curve for 0.8 mg/ml AIPL1 sample exposed for 0.5 s. Superimposed on the experimental SAXS data is the theoretical scattering curve calculated for the AIPL1 model. The linear portion of the Guinier region and the residuals after fit to the experimental data in the low q region (0.558 -1.231 Å Ϫ1 ) such that q⅐R g Ͻ 1.3 are shown in the inset. B, scattering data replotted as a Kratky plot indicating that the sample is mostly globular with some unfolded region. C, scattering data replotted as a Debye-Porod plot approaching the Porod plateau (dotted line) at low angles, indicating that the protein has a folded core. D, pairwise distance distribution function P(r), indicating a multidomain protein with a maximum dimension of approximately 91 Å. AIPL1 contains seven Trp residues, three in each FKBP-like domain and TPR domain and one in the linking region. AMCA-X with a peak absorbance at 350 nm is an excellent acceptor of Trp fluorescence. Thus, in the instance that AIPL1 binds farnesyl-Cys-AMCA, FRET is likely to occur from AIPL1 Trp residues to the fluorescence probe. Indeed, the Trp fluorescence of AIPL1 (excitation at 280 nm) decreased in the presence of farnesyl-Cys-AMCA concomitant with the appearance of peak AMCA fluorescence at 440 nm (Fig. 4A). The binding of farnesyl-Cys-AMCA to AIPL1 was strong (K d ϭ 110 nM) and specific (Fig. 4B). No FRET between AIPL1 and farnesyl-Cys-AMCA was observed in the presence of excess S-farnesyl-Lcysteine methyl ester (data not shown). In addition, using the FRET assay, AIPL1 did not bind Cys-AMCA. Thus, the assay reported primarily, if not exclusively, the interaction of AIPL1 with the farnesyl moiety.
The model of AIPL1 in Fig. 3 shows that the N-terminal FKBP-like domain (residues 1-161) and the C-terminal TPR domain (residues 162-328) are apparently independent with no obvious interdomain contacts. We expressed these domains individually to probe their role in farnesyl binding. Both domains of AIPL1 were expressed robustly in E. coli as soluble proteins (Fig. 4C, inset). Using the FRET binding assay, the FKBP-like domain of AIPL1 bound farnesyl-Cys-AMCA comparably to the full-length protein (Fig. 4, C and D), whereas the TPR domain did not interact with the probe (data not shown). Furthermore, the TPR domain did not alter the affinity of the FKBP-like domain for the probe, when both domains were mixed in the assay (data not shown). Thus, we conclude that farnesyl-Cys-AMCA binds solely to the FKBP-like domain of AIPL1.
Mapping the Farnesyl Binding Site of AIPL1-We first examined the possibility that the farnesyl moiety interaction site of AIPL1 corresponds to the conserved ligand-binding surface of FKPBs. A typical FKBP domain fold comprises a conical fivestranded ␤-sheet surrounding a short ␣-helix (47). Classical FKBPs, such as FKBP12, bind structurally related immunosuppressive drugs, FK506 and rapamycin, within a hydrophobic cavity between the ␤-sheet and the ␣-helix (47). Also, some members of the FKBP family are active peptidylprolylisomerases that catalyze the cis-trans conversion of peptidylprolyl bonds (48). The peptidylprolylisomerase active site overlaps with the FK506-and rapamycin-binding pocket. Although AIP and AIPL1 do not bind FK506 or possess peptidylprolylisomerase activity (30,49), a conserved hydrophobic cavity akin to the ligand-binding site is readily identifiable in the structure of AIP FKBP domain and the model of AIPL1. This site is occluded in AIP, and apparently in AIPL1, by a long insert linking the last two ␤-strands (Fig. 5A) (30). However, the "insert region" appears as a flexible lid that may swing open thereby uncovering the ligand-binding site. Mutations M59A, W72S, and F149A replacing hydrophobic residues of AIPL1 corresponding to prominent ligand contacts in FKBP12 (Phe-46, Trp-59, and Phe-99) (47) were introduced into the full-length mouse AIPL1. Notably, W72S is a mutation found in LCA4 patients (9). An additional P58A mutation was generated as it is predicted to alter the position of M59 as well as the downstream hydrophobic stretch 61 III 63 in the vicinity of the FKBP ligand-binding site. The yields of soluble proteins for all these mutants were similar to that of the wild-type AIPL1 (data not shown). The FRET assay showed no significant differences in farnesyl-Cys-AMCA binding between these AIPL1 mutants and the parent protein ( Table 1).
Examination of the AIPL1 model reveals an unusual hydrophobic patch 147 LVFL 150 at the end of the insert region and the beginning of the last ␤-strand. According to the model, the side chains of Leu-147 and Phe-149 face the interior wall of the ␤-sheet, whereas Val-148 and Leu-150 together with Leu-154 are solvent-exposed on the outside of the ␤-sheet. The F149A mutant was already tested as a part of the FKBP ligand-binding site. Mutants with single Ala substitutions of Leu-147, Val-148, Leu-150, and Leu-154 were generated to probe the potential role of this hydrophobic site in farnesyl-Cys binding by AIPL1. V148A, L150A, and L154A mutants bound farnesyl-Cys-AMCA comparably to AIPL1 (Table 1). Interestingly, the L147A mutation markedly diminished farnesyl-Cys-AMCA binding to AIPL1 (Fig. 5). The expression level of soluble L147A was similar to that of the wild-type AIPL1, arguing against gross misfolding of the protein. The folding of L147A compared with AIPL1 was analyzed by CD spectrometry. The average CD spectra of three scans for AIPL1 and L147A are nearly superimposable (Fig. 5C). The secondary structure composition of AIPL1 and L147A was determined using Selcon 3 and Contin programs within the Dichroweb server (50 -52). The estimated content of 45% helix and 13% ␤-strand for AIPL1 and 46% helix and 13% ␤-strand for L147A are in agreement with the previous CD analysis of AIPL1 (53). Thus, the L147A mutation does not appear to cause any global conformational changes or misfolding in AIPL1.
Farnesyl-Cys Binding by LCA-linked Mutants of AIPL1-Defective farnesyl binding by AIPL1 may hypothetically be the underlying mechanism of certain LCA-linked mutations in the FKBP-like domain. To investigate this hypothesis, we gener- ated the following mutations found as homozygous or compound heterozygous alleles in LCA patients: R38C, K53W (R53W in human AIPL1), V71F, M79T, C89R, V96I, T114I, and Y134F (8 -10, 16). The H82Y mutation (54) was excluded from the analysis as this position is not conserved among species and corresponds to Gly-82 in mouse AIPL1. One of the selected AIPL1 mutants, M79T, failed to express as a soluble protein, suggesting defects in the overall folding. The other AIPL1 mutants expressed well although the yields of soluble proteins varied somewhat (data not shown). The R38C, K53W, V71F, V96I, T114I, and Y134F mutations had no significant effect on the binding of farnesyl-Cys-AMCA to AIPL1 (Table 1). In contrast, the C89R mutation drastically impaired this interaction (Fig. 5). CD analysis of the C89R mutant produced the spectrum similar to that of the wild-type AIPL1 (47% helix and 12% ␤-strand), ruling out global conformational changes in the mutant protein (Fig. 5C). Thus, the C89R mutation locally alters the farnesyl-binding site, or Cys-89 makes direct contacts with farnesyl-Cys-AMCA.
The Insert Region of AIPL1 Is Essential to the Farnesyl Binding-The two mutations disrupting the farnesyl-Cys probe binding to AIPL1, C89R and L147A, flank the insert region (Fig. 5A). To test potential role of this region in the  The estimated secondary structure content of 45% helix and 13% ␤-strand for AIPL1, 47% helix and 12% ␤-strand for C89R, 46% helix and 13% ␤-strand for L147A, and 46% helix and 12% ␤-strand for AIPL1⌬96 -143 is shown.

DISCUSSION
AIPL1 is a specific chaperone for the visual effector enzyme, PDE6. Mutations in AIPL1 cause destabilization of PDE6 and underlie LCA4 (3,8,17,18). In photoreceptor cells, AIPL1 may function as a critical component of a multiprotein chaperone complex involving Hsp90, Hsp70, and other co-chaperones (13). Hsp90/70 proteins interact with the AIPL1 TPR domain, and several LCA-associated mutations within the TPR domain reduced this interaction (13). However, it is not known whether Hsp90/70 proteins directly assist the AIPL1-mediated folding and/or assembly of PDE6. Another well investigated partner of AIPL1 is NUB1 (7,16), but the evidence linking this interaction to PDE6 stability is lacking. Co-expression of PDE6 with AIPL1 in COS cells failed to produce functional PDE6 (18), suggesting the existence of yet additional photoreceptor-specific chaperone(s) for the enzyme.
The mechanism of AIPL1 as a PDE6 chaperone is not fully understood. The PDE6A and PDE6B subunits are prenylated in the cytosol in the rod inner segment. The lipidation promotes association of PDE6A and PDE6B with the endoplasmic reticulum membranes, where the CAAX box is further processed via proteolysis and methylation (27,45,46,55). The timing of PDE6AB heterodimerization in PDE6 biosynthesis remains unclear, but AIPL1 is capable of binding PDE6A prior to the dimer formation and binding of the P␥-subunits (26). Consistent with the idea that AIPL1 binding to PDE6A precedes PDE6AB dimerization, PDE6 subunits appear to misassemble in the absence of AIPL1 (26). The fact that AIPL1 interacts primarily with PDE6A (26), which is farnesylated, and the earlier report of the AIPL1 interaction with farnesylated proteins (28) raised a possibility of direct binding of AIPL1 to the farnesyl moiety. Our study reveals a strong interaction of farnesyl-Cys-AMCA with AIPL1. The binding affinity of farnesyl-Cys-AMCA for AIPL1 is comparable with the probe affinity for the prenyl-binding protein PrBP/␦ (56, 57). The structure of PrBP/␦ complexed with a small G protein Rheb shows that the farnesylated-Cys of Rheb is entirely buried in the hydrophobic pocket of PrBP/␦ (58). Hence, we surmise that the farnesyl moiety is likely to be substantially occluded in the PDE6A complex with AIPL1, and PDE6A would not appreciably interact with the membrane until AIPL1 is released from the complex. Furthermore, the main membrane binding site of PDE6AB is the geranylgeranylated C terminus of PDE6B (59). Thus, our results in the context of the previous studies (26,59) favor the following sequence of AIPL1 interactions during the biosynthesis of PDE6: (i) AIPL1 binds PDE6A after the protein farnesylation in the cytosol, (ii) the binding of AIPL1 allows correct assembly of PDE6A with the geranylgeranylated PDE6B, (iii) assembled PDE6AB is then targeted by the prenylated C terminus of PDE6B to the ER membrane for further processing, and (iv) membrane binding of PDE6AB facilitates release of AIPL1.
Unlike prototypical immunophilin FKBP domains, the FKBP domain of AIPL1 does not bind FK506 or exhibit peptidylprolylisomerase activity (30,49). The FKBP domain may contribute to the AIPL1/NUB1 interaction, but the sites of this interaction are not well defined (16). The role of the AIPL1 FKBP domain, particularly with respect to the function as PDE6 chaperone, remained obscure. A further main finding of our study is the demonstration that farnesylated-Cys binds exclusively to the FKBP domain of AIPL1. The TPR domain of AIPL1 neither promoted nor inhibited the binding of farnesylated-Cys. To map the AIPL1 farnesyl-Cys binding site, we performed mutational analysis of selected residues from candidate hydrophobic areas. Several residues from the hydrophobic pocket corresponding to the conserved FKBP ligand-binding site were found not to be involved in the interaction with the farnesyl-Cys. However, Leu-147 proximal to the FKBP ligand pocket is shown to be critical for the farnesyl-Cys binding. Additional clues to the farnesyl-Cys binding site were provided by the analysis of the LCA-linked mutations of in the AIPL1 FKBP domain. Similar to the L147A mutation, the C89R mutation markedly diminished the interaction of farnesyl-Cys-AMCA with AIPL1. CD analysis of the C89R and L147A mutants rules out protein misfolding or global conformational changes. Cys-89 and Leu-147 are situated in close proximity in the AIPL1 model in a hydrophobic pocket (Fig. 5A). More intriguingly, these residues mark the start and the end of an extensive insert region unique to the FKBP domains of AIP and AIPL1 (30). This indicated a potential role of the insert in farnesyl binding. Indeed, deletion of this region in the AIPL1⌬96 -143 mutant completely abolished farnesyl-Cys-AMCA binding.
The model of AIPL1 (Figs. 3, A and B, and 5A) suggests two interesting features of the insert region that may be important for AIPL1 function. First, this region may influence the relative orientation between the FKBP and TPR domains. The domain orientation in the AIPL1 model is similar to that in FKBP51, and it is rather different in FKBP52 (Fig. 3B). The insert region in AIPL1 would engage in steric clashes with the TPR domain preventing it from adopting the domain orientation seen in FKBP52. Second, this region serves a lid over the wider side of the conical ␤-sheet. Local conformational changes in the hinge sites of the insert region would affect the opening/closing of this lid. Cys-89 and Leu-147 may be essential to the conformation of the insert region, which either provides access to or itself is a part of the lipid-binding site. Our results support the latter model, where the insert region plays a direct and critical role in farnesyl binding. A full understanding of the AIPL1 farnesylbinding site and the selectivity of AIPL1 for the farnesylated PDE6A protein awaits future structural studies.


extfieldsofstudy: Biology Medicine

text:
Structure-Function Studies of the Staphylococcal Methicillin Resistance Antirepressor MecR2*

Background: PBP2a-based methicillin resistance in S. aureus is regulated by the protein MecR2. Results: The structure of MecR2 shows a dimeric multidomain ROK family protein, which nonspecifically binds oligonucleotides but not sugar ligands. Conclusion: MecR2 represents an evolution within ROK proteins to give rise to a protein-binding antirepressor. Significance: The present results pave the way for the design of new antimicrobials. Methicillin resistance in Staphylococcus aureus is elicited by the MecI-MecR1-MecA axis encoded by the mec locus. Recently, MecR2 was also identified as a regulator of mec through binding of the methicillin repressor, MecI. Here we show that plasmid-encoded full-length MecR2 restores resistance in a sensitive S. aureus mecR2 deletion mutant of the resistant strain N315. The crystal structure of MecR2 reveals an N-terminal DNA-binding domain, an intermediate scaffold domain, and a C-terminal dimerization domain that contributes to oligomerization. The protein shows structural similarity to ROK (repressors, open reading frames, and kinases) family proteins, which bind DNA and/or sugar molecules. We found that functional cell-based assays of three point mutants affecting residues participating in sugar binding in ROK proteins had no effect on the resistance phenotype. By contrast, MecR2 bound short double-stranded DNA oligonucleotides nonspecifically, and a deletion mutant affecting the N-terminal DNA-binding domain showed a certain effect on activity, thus contributing to resistance less than the wild-type protein. Similarly, a deletion mutant, in which a flexible segment of intermediate scaffold domain had been replaced by four glycines, significantly reduced MecR2 function, thus indicating that this domain may likewise be required for activity. Taken together, these results provide the structural basis for the activity of a methicillin antirepressor, MecR2, which would sequester MecI away from its cognate promoter region and facilitate its degradation.

reduced MecR2 function, thus indicating that this domain may likewise be required for activity. Taken together, these results provide the structural basis for the activity of a methicillin antirepressor, MecR2, which would sequester MecI away from its cognate promoter region and facilitate its degradation.
Staphylococcus aureus is the most prevalent human infectious agent associated with nosocomial and community infections. It has an extraordinary capacity to become resistant to antibiotics: it was the first bacterial pathogen reported to have become insensitive to penicillin (1)(2)(3)(4). Among the distinct strains is methicillin-resistant S. aureus (MRSA), 5 which currently refers to strains that are generally resistant to ␤-lactam antibiotics (BLAs; penicillins and cephalosporins). Some strains are also resistant to other chemotherapeutics such as aminoglycosides, glycopeptides, macrolides, lincosamides, and fluoroquinolones (3,(5)(6)(7)(8). MRSA is characterized by its ability to thrive in the presence of BLAs because of the biosynthesis of a penicillin-binding protein with low susceptibility to BLAs, termed PBP2a, PBP2Ј, or MecA. The latter is encoded by the gene mecA, which is contained in a transducible mobile element, staphylococcal chromosomal cassette mec (9 -11). Staphylococcal chromosomal cassette mec also includes two genes, mecI and mecR1, which encode a transcriptional DNAbinding repressor, MecI, and an integral membrane zinc-dependent sensor/signal transducer metalloproteinase, MecR1, respectively (12,13). This system is homologous to the blaI-blaR1-blaZ signal transduction system that triggers synthesis of a ␤-lactamase (BlaZ) in both MRSA and methicillin-susceptible S. aureus, as well as in Bacillus licheniformis (14 -16). The currently accepted working model hypothesis for these systems predicts that MecI/BlaI constitutively represses its own biosyn-thesis and that of MecR1/BlaR1 and MecA/BlaZ through binding to the mec/bla promoter (10,13,17). Once MecR1/BlaR1 detects the presence of BLAs through its extracellular sensor domain (18 -22), a signal is transmitted across the membrane to the intracellular zinc-dependent metalloproteinase domain, which becomes activated through proteolytic cleavage (23)(24)(25). This yields functional MecR1/BlaR1, which in turn woulddirectly or indirectly-cause cleavage of MecI/BlaI (26,27). This cleavage would render the dimeric repressor inactive and release it from its DNA-binding site. A similar effect of inhibitor inactivation, which was not based, however, on a proteolytic pathway but on the intracellular presence of a short dipeptidic peptidoglycan fragment induced by BLA stress, has been recently described for B. licheniformis (28). Finally, release of transcriptional repression would elicit biosynthesis of MecA/BlaZ. However, some lines of evidence are not explained by this ternary model. First, the reported cleavage sites of MecR1/ BlaR1 and MecI/BlaI are not compatible with a single proteolytic substrate specificity (29). Second, the structure of MecI and BlaI in complexes with target DNA revealed that the repressor cleavage site is found within an ␣-helix and is not surface-accessible (29 -32), as would be required for proper proteolytic processing. Third, highly resistant MRSA strains did not show significant variation in the phenotypic expression of resistance when wild-type MecI was overexpressed in trans (33). Lastly, the presence of functional MecR1-MecI did not correlate with the level of BLA resistance in a representative collection of epidemic MRSA strains (33). These and other findings led several authors to postulate the existence of a further regulatory element, MecR2/BlaR2, although no candidate molecules were suggested (4, 10, 17, 18, 31, 32, 34 -38).
Most recently, comparative genomic sequence analysis revealed that, in some clinical MRSA strains, a putative gene is found upstream of mecA, which is co-transcribed with mecI and mecR1 (34). Its transcript could be detected by reverse transcriptase PCR of cultures induced with oxacillin-a methicillin analog that has replaced the latter in clinical use-from a set of prototype clinical MRSA strains, paralleling mecA induction. Furthermore, in the presence of fully functional MecR1 and MecI, this gene was essential for the optimal expression of BLA resistance. Finally, in vitro and in vivo assays showed that the encoded protein acted as an antirepressor by disrupting MecI binding to the mecA promoter and fostering its MecR1-independent proteolytic inactivation (34). Collectively, these findings indicated that the long sought-for gene encoding MecR2/ BlaR2 had been found, and so it was termed mecR2 (34).
To shed light on the structural determinants of folding and function of MecR2, we developed an efficient protocol to produce and purify large quantities of the functional wild-type protein. Furthermore, we assayed activity of MecR2 in cell-based assays and in vitro and determined its x-ray crystal structure. We report here the molecular determinants of its function, which were further validated by mutational studies.

EXPERIMENTAL PROCEDURES
Recombinant Overexpression and Purification-The mecR2 gene was amplified from genomic DNA from S. aureus strain HU25 (Met 1 -Ala 376 ; see GenBank TM accession number AF422694, protein sequence identical to UniProt entry Q99XE2) by PCR and cloned into expression vector pCri8a between NcoI and XhoI restriction sites, giving rise to plasmid pCri8::mecR2 (for strains and plasmids used in this study, see Table 1). This construct added an N-terminal His 6 tag and a tobacco etch virus (TEV) protease cleavage site, so that the N terminus of the mature protein was preceded by a 20-residue segment ( Ϫ21 MGSSHHHHHHSSGENLYFQGP Ϫ1 ; negative superscript numbers refer to extra N-terminal residues preceding the  6 tag was assayed under different conditions, but yields were not satisfactory. As such, crystallization trials were performed using tagged MecR2 (hereafter wild-type MecR2). The selenomethionine variant of MecR2 was obtained in the same manner, except that 30 min before induction, the cells were added to 500 ml of medium lacking methionine and containing 25 mg of selenomethionine (Sigma-Aldrich) instead. Circular Dichroism Experiments-The CD spectra of wildtype MecR2 and MecR2 Thr 150 -Ile 160 3 GGGG were recorded with a JASCO J-815 CD spectrometer operated with the following parameters: response, 1 s; scan speed, 50 nm/min; data acquisition interval, 0.1 nm; accumulations, 3; and bandwidth, 1 nm. Quartz cuvettes for far UV (190 -250 nm) with path lengths of 1 mm were used. The samples contained 0.2 mg/ml protein in 10 mM sodium phosphate, 40 mM sodium chloride, pH 7.0. The CD spectrum of cuvette and buffer alone was subtracted from the protein solution spectra.
Cell-based Activity of Recombinant MecR2-To assess the activity of recombinant wild-type MecR2 expressed from vector pCri8a::mecR2 and the aforementioned mutants generated by site-directed mutagenesis, the full respective inserts were cloned into a S. aureus expression vector containing the Pspac IPTG-inducible promoter (pSPT181::spac). Briefly, using flanking primers MR2-3D F1/R1, the insert sequence was amplified using the Phusion high fidelity DNA polymerase (New England Biolabs) and, after digestion with XmaI (New England Biolabs), inserted into the XmaI-linearized pSPT181::spac plasmid using the Rapid DNA dephosphorylation and ligation kit (Roche Applied Science), according to the manufacturer's recommendations. Ligation reactions were transformed into E. coli DH5␣ cells. Recombinant plasmid  JULY 19, 2013 • VOLUME 288 • NUMBER 29 integrity was confirmed by restriction analysis, and the correct insert orientation was confirmed by PCR using primer pairs spacF1/MR2-RT2 and spacR1/MR2-RT1. Insert sequences were also confirmed by DNA sequencing at STAB Vida. After stabilization in E. coli, the recombinant plasmid was electroporated into S. aureus restriction-deficient strain RN4220 and finally transduced by the 80␣ phage to the knock-out mecR2 mutant strain N315 (N315::⌬mecR2), as described previously (41,42). The ability of the recombinant wild-type and mutant MecR2 expressed in trans to complement the N315::⌬mecR2 oxacillin resistance phenotype was then evaluated, as described previously (34).

Molecular Analysis of Methicillin Resistance Factor MecR2
Cross-linking Experiments-Recombinant MecR2 (45.0 kDa) was mixed with MecI (14.8 kDa)-produced as described previously (30)-at a molar ration of 1:2.8 in 50 l of 100 mM HEPES, pH 9.0. Paraformaldehyde was added as cross-linking agent at 0.1% (v/v), and the mixture was incubated at room temperature. The reaction was stopped at distinct time points by adding 10 l of 5ϫ Laemmli buffer with ␤-mercaptoethanol. Samples were analyzed by 10% Tricine SDS-PAGE gels stained with Coomassie Blue. Control experiments were performed with both purified proteins alone under the same experimental conditions.
Western Blotting Analysis-The cross-linking reaction was performed as described above, and three different time points (0, 10, and 30 min) were analyzed in 15% Tris-glycine SDS-PAGE. After electrophoresis, the proteins were transferred to 0.45-m nitrocellulose membranes (Trans-Blot; Bio-Rad), which were blocked at room temperature for 1 h with 20 ml of blocking solution (137 mM sodium chloride, 2.7 mM potassium chloride, 4.3 mM disodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 0.05% Tween 20) with 6% low fat milk. MecI and MecR2 were detected by immunoblot analysis using custom polyclonal antibodies (from Eurogentec) at dilution 1:1000 and a secondary antibody (goat anti-rabbit IgG (HϩL) peroxidase-conjugated antibody; Pierce) at dilution 1:50,000 in 10% blocking solution. The immune complexes were detected using an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent; Pierce) according to the manufacturer's instructions. The membranes were exposed to hyperfilm ECL films (GE Healthcare).
Crystallization and Structure Analysis-Crystallization assays were performed by the sitting drop vapor diffusion method. Reservoir solutions were prepared by a Tecan robot, and 100-nl crystallization drops were dispensed on 96 ϫ 2-well MRC plates (Innovadyne) by a Cartesian nanodrop robot (Genomic Solutions) at the High-Throughput Crystallography Platform at Barcelona Science Park for initial screenings at both 20 and 4°C in Bruker steady temperature crystal farms and using initial protein concentrations of 5.4 and 2.7 mg/ml. Preliminary crystallization hits were improved, and the best conditions were scaled up to the microliter range in 24-well Cryschem crystallization dishes (Hampton Research). Crystals suitable for structure analysis were obtained at 5.4 mg/ml protein concentration in 20 mM Tris⅐HCl, 200 mM sodium chloride, pH 7.4, by using 0.2 M sodium chloride, 20% PEG 1000, 0.1 M potassium dihydrogen phosphate/disodium hydrogen phosphate, pH 6.2, as reservoir solution. The crystals were cryopro-tected with reservoir solution supplemented with 30% glycerol. Crystallization conditions for the selenomethione-derivatized protein were similar to the native ones. Complete diffraction data sets were collected from liquid N 2 flash cryocooled crystals at 100 K (provided by an Oxford Cryosystems 700 series cryostream) at beam lines ID23-1 on an ADSC Quantum Q315r CCD detector and ID29 on a Dectris PILATUS 6M pixel detector, respectively, of the European Synchrotron Radiation Facility (Grenoble, France) within the "Block Allocation Group Barcelona." Crystals were orthorhombic, with two molecules per asymmetric unit. The diffraction data were integrated, scaled, merged, and reduced with programs XDS (43) and SCALA (44) within the CCP4 suite of programs (45) ( Table 3).
The structure of MecR2 was solved by a combination of multiple-wavelength anomalous diffraction with SHELXE/D (46) and fragment search and density modification with ARCIMBOLDO (47) by using two native data sets and two data sets from a selenomethionine-derivatized crystal collected at the selenium absorption peak and the inflection point as determined by a previous XANES scan ( Table 3). The resulting electron density map enabled straightforward tracing of the entire polypeptide chain on a Silicon Graphics Octane2 work station with the program TURBO-Frodo (48). Subsequent crystallographic refinement with BUSTER/TNT (49), which included translation libration screw motion refinement and noncrystallographic symmetry restraints, alternated with manual model building until completion of the model. The latter comprised residues Met 1 -Ala 376 , according to UniProt entry Q99XE2 plus an N-terminal proline resulting from the cloning strategy (termed Pro Ϫ1 ; see above) of molecule A and Asp 3 -Ala 376 of molecule B. Three loop segments were disordered and were thus omitted from the final model: Glu 52 -Ser 58 and Leu 152 -Glu 158 of molecule A and Gly 51 -Pro 63 of molecule B. In addition, 1 phosphate anion, 4 potassium cations, 6 glycerol molecules, and 278 solvent molecules were tentatively assigned (Table 3).
Miscellaneous-The figures were prepared with SETOR (50), CHIMERA (51), and TURBO-Frodo. Structure similarities were determined with DALI (52). Experimental model validation was performed with MolProbity (53) and WHATIF (54). Close contacts (Ͻ4 Å) and interaction surfaces (taken as half of the surface area buried at the complex interface) were calculated with CNS (55), and interface shape complementarity was computed with SC (56) within CCP4 (45). In all cases, a probe radius of 1.4 Å was used. Interdomain flexibility was ascertained with HingeProt employing standard settings (Ref. 57). The final coordinates are available from the Protein Data Bank (PDB; code 4IJA).

Recombinant Overexpression and Purification of MecR2-
Previously, the effects of MecR2 on oxacillin resistance had been studied with a short N-terminally truncated variant of the protein as present in the S. aureus prototype strain N315 (34). Preliminary recombinant overexpression assays in E. coli revealed that this short variant was unstable, and as such, an efficient recombinant overexpression system was developed for full-length MecR2 of S. aureus strain HU25 (GenBank TM accession number AF422694) containing an additional 20-residue N-terminal tag for purification (45.0 kDa; hereafter referred to as wild-type MecR2) by means of expression vector pCri8::mecR2. The protein was folded correctly, eluted as a dimer (see Fig. 2C), and proved suitable for structural and functional studies.
Cell-based Activity of Recombinant MecR2-To assess the activity of full-length wild-type MecR2 (including the tag), the insert of vector pCri8::mecR2 was transferred into S. aureus expression vector pSPT181::spac (containing the IPTG-inducible Pspac promoter) to give recombinant plasmid pSPT::spac-mecR2-3D. This plasmid was then transduced into the S. aureus strain N315 mecR2 deletion mutant (N315::⌬mecR2), and its ability to restore the oxacillin resistance phenotype of parental strain N315 was evaluated. As illustrated in Fig. 1, the phenotype of N315 was fully restored in the presence of the inducer (IPTG), demonstrating that the present full-length wild-type MecR2 variant is biologically active.
MecR2 Binds MecI in Cross-linking Experiments-Previous studies had suggested a direct interaction between MecR2 and MecI in a bacterial two-hybrid system and in electrophoretic shift assays of the binding of MecI to the mecA promoter in the presence of MecR2 (34). In this study, we sought to evaluate the binding of recombinant MecI and wild-type MecR2 proteins in vitro. Control cross-linking experiments with each protein alone indicated concentration-dependent dimerization of both proteins (data not shown), in accordance with their dimeric behavior in solution (see above for MecR2 and (30) for MecI). When performing SDS-PAGE of cross-linking reactions of a mixture of MecI and MecR2 with paraformaldehyde, a time-dependent transition leading to a band migrating at ϳ120 kDa was observed ( Fig. 2A). Western blotting analysis with polyclonal antibodies against both proteins at three different time points of the cross-linking reaction confirmed in the ϳ120-kDa band ( Fig. 2A, black boxes) the presence of both MecI and MecR2 (Fig. 2B), which is consistent with a MecR2 dimer (2 ϫ 45.0 kDa) binding to a MecI dimer (2 ϫ 14.8 kDa).
Overall Structure of MecR2-The crystal structure of MecR2 was determined by a combination of multiple-wavelength anomalous diffraction and ab initio approaches, and two molecules are present in the asymmetric unit of the crystal, monomers A and B (see Table 3 for crystallographic data). The monomer structure reveals an elongated shape of roughly 45 ϫ 60 ϫ 80 Å that is subdivided into three domains: an N-terminal DNA-binding domain (NDD), an intermediate scaffold domain (ISD), and a C-terminal dimerization domain (CDD) (Fig. 3A; the orientation of the left panel is hereafter taken as a reference). NDD (residues Pro Ϫ1 /Asp 3 -His 70 ) starts at the front surface, close to the top of the molecule, and enters a small ␣␤ domain. It consists of three ␣-helices (␣1-␣3) followed by a ␤-ribbon (␤1␤2) whose tip-the loop connecting ␤1 and ␤2 (L␤1␤2)-is disordered (Fig. 3, A and B). These elements con- form to the architecture of a winged helix-turn-helix domain as observed in DNA-binding transcriptional repressors, which generally show disordered ␤-ribbon tips when not bound to operator DNA (58). In the latter, ␣1 and ␣2 contribute to creating a scaffold for correct positioning of helix ␣3. This is the recognition helix that penetrates the major groove of doublestranded DNA, as found in the DNA-binding domains (DBDs) of MecI and BlaI, for example (29 -32).
After strand ␤2, the polypeptide chain enters ISD (residues Leu 71 -Asn 193 ϩ Ser 346 -Ala 376 ), which contains a central twisted five-stranded ␤-sheet (␤3-␤6 plus ␤9) that is parallel for all but one of its strands and shows connectivity Ϫ1, Ϫ1, ϩ3x, ϩ1x (Fig. 3, B and C). On its right, the sheet accommodates two helices (␣4 and ␣5) and a short ␤-hairpin (␤7␤8), which is inserted between ␤6 and ␣5 and is folded back toward the sheet (Fig. 3, A and B); on its left, two perpendicular helices (␣6 and ␣12) are found. In monomer A, segment Leu 152 -Glu 158 within L␤8␣5 on the front surface of the molecule is disordered (Fig. 3, A and B). Inserted between the latter helices is the CDD (residues Leu 194 -Thr 345 ), which starts with a five-stranded ␤-sheet (␤10 -␤12 plus ␤15 and ␤16) that is equivalent to the one found in ISD, both in connectivity and in topology (Fig. 3, B  and C). On its bottom side, this sheet is decorated with helices inserted between ␤12 and ␤15 (␣7-␣9) and between ␤15 and  ␤16 (␣10 and ␣11). In addition, a long ␤-ribbon (␤13␤14) is inserted between ␤12 and ␣7; it contributes to oligomerization (see below). The overall architecture of ISD and CDD is such that the two respective ␤-sheets trap helices ␣6 and ␣12 in between, so that a pseudo-2-fold axis is generated that matches one sheet plus its helix with the other sheet-helix pair (Fig. 3B). Thus, ␣6 could be formally assigned to either ISD or CDD. The interface between these two domains contributes to an apparent ligand-binding cleft (Figs. 3B and 4A). It is framed by L␤6␤7 and ␤7 at its top; ␤10, L␤10␤11, ␤11 plus L␤12␤13 and L␤14␣7 at its bottom; L␤9␣6 and ␣6 at its back; residue Tyr 82 of L␤3␤4 on its right; and Arg 200 of ␤10 and Glu 177 of ␤9 on its left (Fig.  4A). The cleft accommodates two (potential) potassium cations and a (tentative) phosphate anion in monomer A; in monomer B only one cation-binding site is found, which is created by atoms Asn 181 O␦1, Ala 210 O, and Ala 226 O, all 2.6 -2.9Å apart from the metal. A further three (monomer B) and four (monomer A) solvent molecules 3.0 -3.6 Å apart from the metal complete the ligand sphere of this site. The rightmost potassium of monomer A is much more loosely bound, with just two protein atoms at Ͻ3.5 Å: Asn 314 O and Ser 203 O␥. Finally, the phosphate anion of monomer A is bound by Asn 178 N␦2, His 140 N␦1, Glu 179 O⑀1, and a solvent molecule (Fig. 4A).
Oligomeric State of MecR2-MecR2 eluted as a dimer in calibrated size exclusion chromatography, and a dimer was also found to bind a MecI dimer in cross-linking experiments (see FIGURE 3. Molecular structure of MecR2. A, ribbon-type plot of MecR2 in three orthogonal views depicting the NDD (cyan helices and blue strands), ISD (yellow helices and orange strands), and CDD (pink helices and magenta strands). The tentatively assigned potassium and phosphate ions (monomer A) are depicted as green and orange/red spheres, respectively. The flanking residues of the disordered regions within NDD and ISD are indicated by black and gray arrows, respectively. B, topology scheme of MecR2 showing the regular secondary structure elements of MecR2 with their labels and delimiting amino acid positions. A 2-fold axis relating the ␤-sheets of ISD and CDD is depicted in discontinuous trace. The position of the ligand-binding cleft is further indicated. C, cartoon depicting the topology of the main building elements of ISD and CDD, the five-stranded ␤-sheet, and its three flanking helices. Each structural element carries the labels as found in the two domains. The arrows hallmark insertion points of distinct secondary structure elements within each domain: encircled 1 for ISD and encircled 2 for CDD. above). Consistent with this, the two molecules found in the crystal asymmetric unit give rise to a dimer with a large interaction surface (1,465 Å 2 ; ϳ8% of the total surface of a monomer) with complementarity (0.72) that is in the range reported for protein oligomers and protein/protein inhibitor interfaces (0.70 -0.76) (56). This interaction includes 83 contacts (Ͻ4 Å), among them roughly symmetric hydrophobic contacts between nine residues of either monomer and 34 hydrogen bonds and polar interactions. Altogether, these findings point to biological relevance for the dimeric arrangement. Protein segments involved in dimerization are provided by each CDD: L␣6␤10, L␤11␤12, the second half of ␣9 and L␣9␤15, and ␤-ribbon ␤13␤14. The two monomers are not completely equivalent, and this gives rise to an root mean square deviation value upon superposition of 0.97 Å for 353 C␣ atoms deviating less than 3 Å out of 361 common residues. Analysis of interdomain flexibility based on the elastic network model revealed potential hinge motions at the two domain junctions of each monomer, which increase upon going from the CDDs to the NDDs (Fig. 4C).
Structural Similarities-Sequence similarity searches suggested that MecR2 groups with the ROK family of proteins (from repressors, open reading frames, and kinases), which includes transcriptional repressors and sugar kinases (59 -61).
One archetypal ROK protein is xylose transcriptional repressor (XylR), which regulates xylose utilization as a carbon source in bacteria (62)(63)(64)(65)(66). However, there is no structural data on XylR available. E. coli protein Mlc is the only functionally and structurally characterized ROK family protein with DNA repressor function (60,67). Mlc is a dimeric/tetrameric transcriptional repressor that controls the utilization of glucose in E. coli (68). It shows overall fold similarity and quaternary arrangement with MecR2 and is likewise subdivided into three domains equivalent to NDD, ISD, and CDD. In addition, two unpublished structures corresponding to proteins of unknown function from Thermotoga maritima (PDB code 2HOE) and Vibrio cholerae (PDB 1Z05), deposited with the PDB by structural genomics consortia, also displayed high structural similarity scores with MecR2. These are the only three-domain ROK proteins structurally reported, which form part of a large group of mostly two-domain (ISDϩCDD) ROK proteins, generally dimeric or tetrameric sugar kinases that bind and phosphorylate glucides (59 -61, 69).
MecR2 Has a Nonfunctional Ligand-binding Cleft-As for MecR2, Mlc has a ligand-binding cleft that sits at the interface between ISD and CDD. It further has an adjacent regulatory zinc-binding site, which is required for repressor activity (60) and is provided by the segment topologically equivalent to the protruding ␤-ribbon ␤13␤14 in MecR2. In contrast to the latter, however, this segment is folded back toward the body of the molecule in Mlc, in a fashion similar to that in ROK glucokinase from E. coli (70) and glucomannokinase from Arthrobacter sp. (71), where it contributes to shaping the floor of a sugar-binding cleft. This segment encompasses a widely conserved consensus sequence among ROK proteins, CXCGXXGCXE (60,69), which contains three zinc-binding cysteine residues. A similar site is also found in Bacillus subtilis fructokinase YdhR (72), an undescribed putative glucokinase from Enterococcus faecalis (PDB code 2QM1), an undescribed putative regulatory protein from Salmonella typhimurium (PDB code 2AP1), and the aforementioned protein from V. cholerae, so that ROK family members containing this consensus sequence share a conserved metal-binding site. By contrast, MecR2 lacks these cysteine residues, and its chain trace is completely different in the corresponding region, giving rise to an extended ␤-ribbon that engages in dimerization (see above). This ␤-ribbon is similar in the aforementioned protein from T. maritima, although in this case the ribbon is four residues shorter than that in MecR2. Only the last glutamate of the consensus sequence is found in the latter two proteins-Glu 248 in MecR2-and it contributes to the hypothetical ligand-binding cleft (see above).
Another ROK signature motif is found in several ROK proteins comprising the C-terminal residues EXGH, about 10 residues upstream of the previous consensus sequence (see Fig. 4 in Ref. 69). The histidine-missing in MecR2-is engaged in zinc binding in Mlc and the V. cholerae protein, whereas the glutamate-equivalent to Glu 228 in MecR2-is engaged in sugar binding in E. coli glucokinase and Arthrobacter sp. glucomannokinase, together with the conserved residues at positions equivalent to Glu 248 , Asn 178 , and Glu 179 in MecR2. The latter two residues are engaged in phosphate anion binding (see above). Although these residues are likewise conserved in Mlc, this protein does not bind glucose, i.e., its regulation does not depend on allosteric changes induced by sugar binding (73): inactivation is exerted through recruitment by the glucose transporter protein EIICB Glc of the phosphotransferase system (74 -76). By contrast, XylR binds xylose, glucose, and glucose-6-phosphate in vitro (66), i.e., it is a three-domain transcriptional repressor with a functional regulatory sugar-binding cleft. Overall, these findings indicate that ROK proteins include members that bind sugars such as the sugar kinases and XylR but also some that do not such as Mlc. Accordingly, we set out to assess whether MecR2 has a functional sugar-binding ligandbinding site despite lacking the zinc-binding site, and three mutants affecting participating residues (MecR2-Asn 178 -Glu 179 3 Ala-Ala, MecR2-Glu 228 3 Ala, and MecR2-Glu 248 3 Ala) were constructed and assayed for their functional roles in the mec locus. These studies revealed that the mutants showed antirepressor activity in cell-based assays that was indistinguishable from the wild-type protein (Fig. 1). We therefore conclude that ligand binding is not required for function in MecR2, i.e., that it has a nonfunctional ligand-binding cleft.
MecR2 Has Nonspecific DNA Binding Capacity-Three-domain ROK transcriptional repressors such as Mlc and XylR possess N-terminal DBDs that engage in DNA-operator binding and thus in the regulation of the transcription of the respec-tive effector genes (60,65). MecR2 NDD likewise conforms to the structural determinants of such a DBD (see above). The reported structures of Mlc, T. maritima, and V. cholerae are DNA-unbound, and they display the two recognition helices of a dimer in a relative spatial arrangement that is not adequate for binding to two successive turns of the major groove of dsDNA (60,67). This is consistent with the finding that structural flexibility-which allows for major structural rearrangement-of Mlc was identified as essential for DNA binding activity and regulatory function (67).
We set out to assess the DNA binding capacity of MecR2 in an electrophoretic mobility shift assay in the presence of a 25-bp dsDNA encompassing the Z-dyad sequence of the bla promoter sequence (MR2-EMSA1 and 2 in Table 2), which had been used for structural and functional studies of MecI (29,30). We found that MecR2 strongly bound and completely retarded this DNA at a protein:dsDNA ratio of 4:1 (Fig. 5, left panel). Similar effects were observed on another 25-bp dsDNA of scrambled sequence (see MR2-EMSA 3 and 4 in Table 2; Fig. 5, right panel). These results indicate strong but unspecific DNA binding in vitro. To assess the potential biological importance of this function, a deletion mutant was constructed, MecR2-⌬Ser 55 -Lys 62 , in which seven residues of L␤1␤2 within the winged helix NDD had been deleted. This variant showed significantly diminished antirepressor activity when compared with the wild-type protein (Fig. 1). We conclude that MecR2 possesses a functional DBD that may be required for exerting oxacillin resistance.
ISD May Be Relevant for Function-In the search for biologically relevant regions of the structure of MecR2, we noticed that the flexible segment contained within L␤8␣5 of ISD was located on the surface of one of the two monomers within the dimer (see above and Fig. 4B). We constructed a mutant in which an 11-residue stretch was replaced by 4 glycines (MecR2-Thr 150 -Ile 160 3 GGGG) to maintain the overall structure of the domain and assayed its cell-based activity (Fig. 1). Similarly to MecR2-⌬Ser 55 -Lys 62 , this variant was not capable of restor- ing the oxacillin resistance phenotype in the presence of inducer.
To verify that this effect was not due to an unfolded protein variant, MecR2-Thr 150 -Ile 160 3 GGGG was recombinantly overexpressed under the same conditions as wild-type MecR2. Both proteins evinced comparable elution profiles in calibrated size exclusion chromatography, which revealed that both proteins were well folded and dimeric (Fig. 2C). In addition, circular dichroism experiments showed indistinguishable spectra for both protein variants, which likewise correspond to properly folded proteins (Fig. 2D). Accordingly, the phenotype observed for the mutant is actually due to the missing ISD fragment, and we conclude that this region may be implicated in biological activity.
Functional Implications of MecR2-The similarity of MecR2 with ROK family bacterial sugar kinases and transcriptional repressors, both in the overall monomeric structures and in the general dimeric quaternary arrangements, have evolutionary and functional implications. Accordingly, XylR would represent the first step in evolution from an ancient two-domain ROK sugar-binding kinase-putatively evolved from a common ancestral hexokinase (69, 77)-refurbished to produce a three-domain DNA-binding transcriptional repressor through N-terminal fusion with a winged helix-turn-helix DBD. XylR still binds and is allosterically regulated by sugar. Mlc would represent the next step-as already anticipated in (60)-to a three-domain DNA-binding transcriptional repressor that does not bind sugar and is not regulated by binding to an inducer or by proteolytic cleavage but through sequestration by a glucose transporter, i.e., through a protein-protein interaction (67,76). Finally, MecR2 would represent a last step in the evolutionary process, in which a three-domain ligand-independent Mlc-like repressor would have kept an unspecific DNA binding ability putatively required for biological function as an antirepressor. In addition, the dimeric protein would have developed a region within its ISD that potentially would likewise be required for antibiotic resistance. This antirepressor activity would entail binding and sequestering away from its cognate promoter a dimeric transcriptional repressor, MecI. Finally, MecI sequestering would suppress its repressor activity of the mec locus by promoting its proteolytic cleavage, presumably by native proteinases, and enhancing the signal transduction mediated by the cognate integral membrane sensor-transducer, MecR1. This, in turn, would trigger the methicillin resistance response.


s2fieldsofstudy: Biology Medicine

text:
HLA class II SNP interactions and the association with type 1 diabetes mellitus in Bengali speaking patients of Eastern India

Background Several studies have demonstrated a fundamental role for the HLA in the susceptibility of, or protection to, type 1 diabetes mellitus (T1DM). However, this has not been adequately studied in Asian Indian populations. To assess the frequency of HLA class II (DPA1, DPB1, DQA1, DQB1 and DRB1) associated to susceptibility or protection toT1DM in a Bengali population of India with diabetes. Results Single nucleotide polymorphism study. The HLA genotyping was performed by a polymerase chain reaction followed by their HLA-DP, DQ, and DRB1 genotypes and haplotypes by sequencing method. The results are studied by Plink software. The χ2 tests were used for the inferential statistics. To our knowledge, this study is the first of a kind which has attempted to check the HLA association with T1DM by SNPs analysis. The study recruited 151 patients with T1DM and same number of ethno-linguistic, sex matched non-diabetic controls. The present study found a significant SNP rs7990 of HLA-DQA1 (p = 0.009) negative correlation, again indicating that risk from HLA is considerably more with T1DM. Conclusions This study demonstrates that the HLA class-II alleles play a major role in genetic basis of T1DM.

Background
Type 1 diabetes mellitus (T1DM) (OMIM-222100), results from a cellular-mediated autoimmune destruction of the Beta-cell of the pancreas [1]. T1DM is a disease of major public health concern [2][3][4]. The previous studies showed that in India, the prevalence of T1DM varies from about 1.6 to 10.5/100000/year [5,6]. The epidemiological study conducted in South Indian population for four years, suggested the prevalence of T1DM in India is increasing. The overall prevalence of T1DM in Karnal district, a North Indian city with a population of 222017, is 10.20/100,000 population [7].
The genetic risk factors of T1DM are better understood than the environmental risk factors. Studies on Human and animal models show the MHC class II-mediated effects on the disease susceptibility [8]. Early studies identified in the Human Leukocyte Antigen (HLA) genes, located on chromosome 6p21.31 as T1DM susceptibility genes. Resulting studies showed an association between the insulin gene on chromosome 11p15.5. The risk of T1DM is linked with about 18 regions of the genome. These regions, each of which may contain multiple genes, labeled IDDM1 to IDDM18. The best studied is IDDM1, which contains the HLA genes encode proteins of the immune response [9,10].
HLA is one of the most polymorphic genetic systems in Human genome. IMGT/HLA database have reported 6,275 HLA alleles [11]. Because the HLA class II molecules are polymorphic, they can embrace a wide variety of antigens in their antigen-binding groove and present them to diverse T-lymphocyte antigen receptors, triggering antigen recognition. Several studies have displayed HLA class II alleles, DQ and DR influence T1DM susceptibility. The contribution of the DQ molecules to overall disease susceptibility might be genotype dependent and/or may be influenced by the DRB1*04 allele on the haplotype [12].
The role of DP molecules has yet to be resolved satisfactorily. The results favor DPB1*0301 and DPB1*0202 alleles as predisposing for T1DM [24]. Analysis of family-based data from the Human Biological Data Interchange (HBDI) repository and Italian studies, suggests the presence of a T1DM protective locus at or near DPB1*0101 [25]. It is hypothesized that the strongest candidates for increasing T1DM risk among DR3-DQB1*0201/DR4-DQB1*0302 individuals is of alleles of DP and DRB1*04 subtypes and, in particular, the absence of reportedly protective alleles DPB1*0402 and/or DRB1*0403 [26].
There are some studies on HLA-DP in different ethnic groups mainly about HLA-DPB1. In a study from Sudan, there were no significant differences between Sudanese patient and control groups in HLA-DPB1 frequencies [27]. Although, there was also no noticeable association between T1DM and HLA-DPB1 allele in Japanese [28]. From Indian T1DM patients, so far no studies have been reported on DP molecules.
Although different methods exist to characterize the polymorphisms in HLA genes, the 12 th International Histocompatibility Workshop suggested the Sequence Based Typing (SBT) methods [29]. Therefore, we conducted a hospital-based case-control study in the West Bengal region of India to find out the role of HLA-DRB1, DQ and DP gene polymorphisms in progress T1DM.

Subjects
In the present study 151 T1DM patients were recruited from six different hospitals -Calcutta Heart and Research Clinic; Endocrinology Department, Calcutta Medical College & Hospital; Endocrinology Department, SSKM Hospital; Netaji Subhash Chandra Bose Cancer Research Institute; Rabindranath Research Institute of Cardiac Sciences; School of Tropical Medicine; from metropolitan Kolkata. The inclusion criteria considered for recruitment of cases were an onset of diabetes below 39 years of age, and presenting with or without acute ketosis with absolute insulin dependence, as shown by a deficient C-peptide secretion i.e., a C-peptide value less than 0.6 (0.6-3.2)ng/ml and antibody positivity for Glutamic Acid Decarboxylase antigen(GADA) and, Insulinoma-Associated Protein-2 Antibodies (/IA-2/ICA512) [30]. Patients of at least one year duration were selected to exclude acute or "honeymoon" phases.
The sampled subjects speak Bengali. The samples represented in our study is mainly originated from districts-Kolkata, followed by South-24-Parganas, North24-Parganas, Howrah, Hoogly, comprising a small geographical area and historically forming a cultural zone. West Bengal is the melting pot of Indo-Aryan, Austric, Dravidian, Tibeto-Burman and various other languages [31]. Some scholar stated that Bengal had many striking resemblance with the Dravidian culture [32], where as others suggested that Bengal as the meeting place of Aryan, non-Aryan, and Mongoloid races [33]. The controls used in the present study represent 151 healthy individuals without T1DM and T2DM in the family history, and matched for ethnicity, geography and socioeconomic status and higher age compared to cases. Blood samples (3 ml) were collected into EDTA-coated vacutainers from both cases and controls with written informed consent. This research was approved by the Institutional Review Board of the Anthropological Survey of India as well as by the respective hospital's ethical committee.
Genotyping DNA was isolated according to the standard protocol [34]. Genotyping of the following HLA genes HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1 was performed using PCR followed by sequencing. The primers used in the present study to amplify different regions of aforementioned genes are documented in Table 1. A total volume of 10 μl was used for each PCR reaction which were carried out in an ABI Gene Amp PCR system 9700. The nucleotide sequences of the PCR products were determined by direct sequencing using di-deoxy chain terminator cycle sequencing protocol through 3730 DNA Analyzer (BigDye V3.1, Applied Biosystems; Foster City, CA, USA). Sequencing was carried out with both the forward and the reverse directions. Problems in genotype assignment for certain samples on the DRB1 and the DQB1 loci that aroused because of the genotypic ambiguity were surmounted by following the guidelines of the American Society for Histocompatibility and Immunogenetics [35].

Statistical analysis
All nucleotide changes detected by using SeqScape software v 2.5 (Applied Biosystems) and with the wild gene using pair-wise BLAST [36]. Class II HLA genes are more polymorphic compared to other genes and thus the editing was much more complicated for Class II HLA. Allele and genotype frequencies of the SNP data compared between T1DM patients and healthy control groups. Statistical analysis for HWE was performed by Plink v 1.07 software [37]. Evaluation of the genotype or allele frequencies of cases and controls was carried out by calculating the odds ratios (OR) with 95% of confidence intervals (CI). A P value less than 0.05 were considered as statistically significant. Haplotype frequencies and linkage disequilibrium estimated by using Haploview v 4.1, which measures D' and r 2 between each pair of SNPs and to define haploblocks [38].

Results and discussions
Exonic 331 SNPs in the Class II HLA-DP-DQ-DRB1, identified and tested for HWE by Plink software. Only ten SNPs found to be in equilibrium for control, only these 10 SNPs were used in further analysis. Of the 10 SNPs, eight were from HLA-DQA1 and the remaining were from HLA-DPA1 (rs2308911, rs2308912) ( Table 2). Allele and genotype frequencies of all the SNPs for both cases and controls were presented in Table 2. Except for rs7990 (p = 0.009), there were no significant differences in genotype or allele frequencies of these SNPs between controls and T1DM cases (Table 2). Thus, after all prune and excluding from 331 SNPs, rs7990 SNPs of HLA-DQA1 shows a negative association with T1DM. The pairwise LD values (D' and r2) among studied SNPs were provided in Figure 1 and in Table 3. Linkage Figure 1 Pair-wise linkage disequilibrium between the ten SNP markers in HLA-DQA1-DPA1 genes. disequilibrium analysis had revealed strong LD and formed 3 haplotype blocks, suggested that haplotype study might be useful. Haplotype-phenotype association analysis using SNPs that were located in the LD blocks in block 1 (rs1047993 and rs707949) showed association with T1DM (Table 3).

Conclusions
The complex nature of the HLA region on chromosome 6p21.31, with the high LD between genes, has made it enormously difficult to explicate the effect of individual genes for the risk of developing T1DM. The studies suggested that HLA Class II DRB1-DQB1 contribute to T1DM susceptibility. Calculating for the influence of class II DR-DQ haplotype and genotype effects, a role in T1DM has been shown of additional HLA Class II DPB1 [16]. New studies by sequencing has yielded many SNPs that include thirteen SNPs from class III alleles, which showed evidence of an effect on T1DM risk, although some of the SNPs are in tight LD with each other. The strongest association within class III markers was with rs2395106 that maps 5 0 to the NOTCH4 gene and the second association was with rs707915 mapping to the MSH5 gene, in a block of six markers significantly associated with T1DM after adjusting for LD with DR-DQ [39].
A widespread SNP analysis of the extended MHC in 237 families with Type 1A from the U.S. and 1,240 families from the T1DGC was conducted and showed an association with Type 1A diabetes (rs1233478, p = 1.6 x 10 -23 ), in the UBD/MAS1L region, telomeric of the classic MHC [40]. Another study from T1DGC on HLA markers showed 296 significant SNPs in a narrow genomic region, some of these markers are close to one another and in strong LD. Therefore, although the SNPs that stand for independent signals without LD, some high-LD markers can produce correlated associations in the study. However, high-LD and long haplotype blocks will also deter fine mapping precisely. This study also shows that SNPs with the smallest P values is from the HLA-DR and -DQ region which confers the major genetic risks for T1DM [41].
Most of these recent studies have used data produced by the T1DGC and therefore are from Caucasoid population, and they conducted the most detailed investigation of the HLA complex in disease, characterizing over 3,000 SNPs, and independently tested all previously reported T1DM susceptibility genes [42,43].
In this study we used a sequencing SNP approach to characterize the polymorphisms in HLA genes. We carried out sequencing in 151 cases and 151 normal healthy participants, followed by statistical analysis of the SNPs. Out of 331 exonic SNPs identified, only 10 is following HWE. Thus, after all pruning and excluding from 331 SNPs, and rs7990 SNP of HLA-DQA1 showed significant protection from T1DM. Linkage disequilibrium analysis revealed 3 haplotype blocks and further haplotype-phenotype association analysis did show association between haplotypes in block 1 (rs1047993 and rs707949) and T1DM.
We have also done the study based on alleles and the findings are similar like the DQA1*0103 allele is a novel allele with a significant association with the protection from T1DM in Eastern Indian Bengali population from India [44]. To our knowledge, this study is the first of a kind which has tried to check the HLA association with T1DM by SNPs analysis in India. As said before most of these recent studies have used data formed by the T1DGC, and they use refined statistical methods to control for the complexity of the HLA region because of the extended LD and polymorphic loci. Still the deviation of some results suggests the difficulty of examine the independent genetic contribution of genes in this region to the risk of developing T1DM. It is most likely that more SNPs with individual but smaller or rarer effects on diabetes risk can be identified in this region. However, to find these SNPs new approaches for analyzing genetic association data are needed. In addition, large numbers of subjects may help to give more robust and confident association with T1DM.

Competing interest
There are no conflicts of interest.
Authors' contributions OR carried out the molecular genetic studies, participated in the sequence alignment, did the statistical calculation and drafted the manuscript. BS corrected the manuscript. BLVKS conducted the Thesia calculation. VP suggested the project GS suggested the project SC provided with the samples PR provided with the samples VRR guided the total project and corrected the MS. All authors read and approved the final manuscript.