Solution Binding and Structural Analyses Reveal Potential Multidrug

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Solution Binding and Structural Analyses Reveal Potential Multidrug Resistance Functions for SAV2435 and CTR107 and Other GyrI-like Proteins Andrew Moreno, John R. Froehlig, Sharrol Bachas, Drew Gunio, Teressa Alexander, Aaron Vanya, and Herschel Wade* Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, United States S Supporting Information *

ABSTRACT: Multidrug resistance (MDR) refers to the acquired ability of cells to tolerate a broad range of toxic compounds. One mechanism cells employ is to increase the level of expression of efflux pumps for the expulsion of xenobiotics. A key feature uniting efflux-related mechanisms is multidrug (MD) recognition, either by efflux pumps themselves or by their transcriptional regulators. However, models describing MD binding by MDR effectors are incomplete, underscoring the importance of studies focused on the recognition elements and key motifs that dictate polyspecific binding. One such motif is the GyrI-like domain, which is found in several MDR proteins and is postulated to have been adapted for small-molecule binding and signaling. Here we report the solution binding properties and crystal structures of two proteins containing GyrI-like domains, SAV2435 and CTR107, bound to various ligands. Furthermore, we provide a comparison with deposited crystal structures of GyrI-like proteins, revealing key features of GyrI-like domains that not only support polyspecific binding but also are conserved among GyrI-like domains. Together, our studies suggest that GyrI-like domains perform evolutionarily conserved functions connected to multidrug binding and highlight the utility of these types of studies for elucidating mechanisms of MDR.

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efflux pumps combine with soluble drug sensors to offer cells effective chemoprotective responses that both detect and selectively remove cytotoxic compounds from the interiors of prokaryotic and eukaryotic cells.16,17 MD recognition is central to efflux-related mechanisms that offer broad cellular protection against toxic chemical agents.8,18−21 Current models describing MD recognition by MDR proteins draw from investigations of interactions of ligands with efflux pumps and regulator proteins.22−29 In these studies, structural and other biochemical approaches were combined with diverse sets of chemical probes to identify putative MD-binding pockets as well as structural features within those pockets that confer polyspecific binding.22,30 Thus, these studies provide a framework for distinguishing proteins with promiscuous binding specificity from their highly selective counterparts. Global domain architecture and local features are both functional components of ligand recognition, the latter of which appear to be shared more among MDR proteins than among the former.22 Overall, MD-binding pockets provide

ultidrug (MD) and xenobiotic efflux pumps protect cells by actively removing intracellular pools of toxic compounds.1 Low levels of efflux are chemoprotective in normal functioning cells, and efflux pumps are universally expressed in both eukaryotes and bacteria.2 However, high efflux levels extend protection to drug-targeted cells, rendering them resistant to a broad range of unrelated compounds.3,4 Indeed, an elevated level of expression of efflux pumps has been established as a major contributor to multidrug resistance (MDR) in a growing list of pathogenic bacteria, fungi, and parasites.5−7 For higher eukaryotes, drug efflux is a critical problem in cancer therapy, leading to decreased efficacy of chemotherapeutics and, in many cases, chemotherapy failure and relapse.8,9 In humans, three ATP-binding cassette transporters are known to play significant roles, including Pglycoprotein,10 multidrug resistance-associated protein (MRP1),11 and breast cancer resistance protein (BCRP).12,13 Cells utilize both direct and indirect mechanisms to regulate active chemical efflux.5 Five families of energy-dependent pumps offer direct control by transporting compounds uphill across cell membranes.14 On the other hand, transcriptional regulator and other ligand-responsive signaling proteins, including quorum sensing and two-component systems, indirectly affect drug efflux by controlling efflux pump expression.15 In many cells, © XXXX American Chemical Society

Received: June 27, 2016 Revised: August 4, 2016

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DOI: 10.1021/acs.biochem.6b00651 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

to BmrR, three other proteins harboring GyrI-like domains have been implicated in MDR. In Escherichia coli, the Rob protein is postulated as a global regulator of MDR that contains a C-terminal GyrI-like domain capable of binding small molecules; however, its biological function has yet to be determined.36−39 The other two include GyrI, a proposed protein gyrase inhibitor in E. coli, and Cass2, a protein encoded on an integron gene cassette in Vibrio cholerae.40,41 In the case of Cass2, crystallographic, genetic, and solution binding analyses support a ligand-dependent regulatory function that responds to unrelated cationic compounds. Structural similarities between these proteins and BmrR agree with such a hypothesis. A DALI (distance matrix alignment) search for structural homologues of the C-terminal BmrR domain yielded a number of matches.42 In addition to Rob (Figure 1B), GyrI (Figure 1C), and Cass2 (Figure 1D) described above, matches included three uncharacterized proteins: SAV2435 from Staphylococcus aureus [PDB entry 3LUR, Joint Center for Structural Genomics (JCSG)], CTR107 from Chlorobium tepidum [PDB entry 3E0H, Northeast Structural Genomics Consortium (NESG)], and LIN2189 from Listeria innocua [PDB entry 3B49, Midwest Center for Structural Genomics (MCSG)]. None of these proteins have been characterized by biochemical methods. Intriguingly, these proteins have been annotated as putative MDR regulators. To gain insight into the ligand recognition properties of GyrI-like proteins, we have examined the solution binding properties and determined multiple X-ray structures of SAV2435 and CTR107 bound to various ligands. For SAV2435, we report five structures, including the ligand-free protein (2.0 Å), as well as SAV2435 bound to rhodamine 6G (RH6G) (1.95 Å), tetraphenylphosphonium (TPP) (2.1 Å), and ethidium (ET) (2.10 Å). The set of structures also includes a two-ligand, ternary complex with RH6G and TPP (1.86 Å). Furthermore, we have determined the structure of CTR107 bound to RH6G to 2.00 Å resolution. Crystallographic analyses of SAV2435 and CTR107 as well as structural comparisons against other characterized proteins containing GyrI-like domains, including LIN2189, support a general ligand binding function and, possibly, a signaling function for the GyrI superfamily. Similar to Cass2, BmrR, and, possibly, Rob, both SAV2435 and CTR107 appear to be capable of recognizing multiple, unrelated ligands with appreciable affinities. Moreover, we observe a number of ligand pocket features that appear to be conserved across the family of GyrI-like proteins with known structures. Solution binding results support the crystallographic results. Finally, analysis of the ternary SAV2435 complex with RH6G and TPP reveals in addition to a putative ligand pocket, an alternative hydrophobic protein−protein interaction site that may offer signaling functionality to SAV2435 and other single-domain GyrI-like proteins. Together, these results reveal general features of GyrIlike domains that support polyspecific binding and suggest an evolutionarily conserved function for GyrI-like domains.

adaptable and versatile platforms for forming favorable van der Waals, polar, and electrostatic interactions with chemically diverse ligands.31 Our current views of MD recognition contrast starkly with descriptions of ligand-specific binding, which show high ligand−receptor complementarity and well-defined binding determinants. Because MDR proteins bind to structurally diverse ligands, a unique set of contacts is observed within the binding pocket of each complex. As a result, elucidating recognition elements, key motifs that dictate polyspecific binding, and formulating universal binding models remain significant challenges. BmrR, a MDR gene regulator from Bacillus subtilis, is regarded as a prototype for investigating MD recognition.23,32,33 The MerR-related protein is composed of three domains, including a DNA-binding motif (PFAM entry MerR_1, PF13411), a coiled-coil region that mediates dimerization, and an effector-binding domain (GyrI-like, PF06445)34 (Figure 1A). In contrast to other well-studied

Figure 1. Representative domain architectures of GyrI-like proteins. Crystal structure of (A) BmrR (PDB entry 3Q1M), (B) Rob (PDB entry 1D5Y), (C) GyrI (PDB entry 1JYH), and (D) Cass2 (PDB entry 3GK6). GyrI-like domains are colored dark gray, dimerization domains magenta, and DNA-binding domains dark blue.

canonical MDR proteins, structural and molecular docking studies indicate that BmrR utilizes a small rigid pocket for MD recognition.22 Within the binding site, several well-defined recognition determinants appear to dominate binding for all examined ligands. These include a docking platform composed of six aromatic residues, a hydrophobic residue pair that clamps ligands onto the platform, four pseudosymmetrically disposed, carboxylate-containing residues that offer broad selectivity for structurally unrelated cationic ligands, and a rigid solvation shell composed of a network of ligand-accessible hydrogen-bonding donor and acceptor atoms that line the MD-binding pocket.23,26 The identification of well-defined recognition determinants has not been reported for any other MDR protein studied to date. As such, BmrR provides a structural framework for elucidating similar elements in other MDR proteins. The GyrI-like domain, which interacts with diverse ligands in BmrR, represents a logical initial point for exploring the generality of the recognition determinants elucidated in previous studies of the MerR-related transcriptional regulator.23,26,27,33 On the basis of protein sequence and structural analyses, this domain has already been postulated to have been adapted for small-molecule binding and signaling.35 In addition

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MATERIALS AND METHODS Protein Expression and Purification. S. aureus genomic DNA was a gift from M. Shirtliff (University of Maryland, Baltimore, Baltimore, MD). The sav2435 gene was amplified by polymerase chain reaction using gene-specific primers and inserted into the pET28b+ vector (KanR) to yield the His6tagged variant, which was subsequently transformed into E. coli BL21(DE3) cells. Cells were grown at 37 °C in LB medium, B

DOI: 10.1021/acs.biochem.6b00651 Biochemistry XXXX, XXX, XXX−XXX

C

a

96.97 (160) 3.03 (5) 0.00 (0)

0.019 1.635 1292 33 113

0.013 1.337 1306 0 135

98.18 (163) 1.82 (2) 0.00 (0)

36.67−1.95 (2.02−1.95) 12837 18.71/16.19

73.3, 73.3, 65.3 90, 90, 90 7.5 0.998 (0.513) 23.8 (2.2) 99.87 (99.87) 7.7 (4.9)

73.7, 73.7, 68.4 90, 90, 90 14.9 0.987 (0.787) 13.9 (1.3) 99.91 (99.9) 11.8 (7.9)

36.84−2.00 (2.07−2.00) 13259 20.45/17.69

P41212

P41212

SAV2435 with R6G

98.18 (163) 1.82 (2) 0.00 (0)

0.007 1.155 1289 25 132

73.5, 73.5, 66.4 90, 90, 90 11.7 0.989 (0.902) 17.3 (1.9) 99.94 (99.38) 10.9 (6.0) Refinement 26.00−2.10 (2.31−2.10) 10543 18.99/17.81

P41212

SAV2435 with TPP

96 (165) 0.00 (0) 0.61 (1)

0.003 0.79 1317 39 78

32.46−2.101 (2.176−2.101) 11441 23.52/21.53

73.72, 73.72, 68.53 90, 90, 90 1.6 1 (0.888) 38.04 (4.56) 99.50 (95.15) 2.0 (2.0)

P41212

SAV2435 with ET

96.36 (159) 3.03 (5) 0.61 (1)

0.005 0.99 1300 58 131

36.86−1.86 (1.93−1.86) 15185 19.26/17.64

73.7, 73.7, 65.9 90, 90, 90 13.5 0.999 (0.505) 19.4 (1.5) 96.28 (71.60) 11.6 (2.7)

P41212

SAV2435 with R6G·TPP

98.40 (308) 1.28 (5) 0.32 (1)

0.014 1.33 2373 66 248

47.52−2.00 (2.07−2.00) 27525 17.93/14.15

72.0, 72.0, 146.9 90, 90, 120 7.6 0.996 (0.554) 23.76 (5.66) 94.93 (97.41) 4.4 (4.4)

P65

CTR with R6G

Values in parentheses correspond to data from the highest-resolution shell. bSolvent includes ordered water and glycerol molecules. cValues in parentheses correspond to the number of residues.

resolution (Å) [I/σ(I) = 1.0]a no. of unique reflections Rfree/Rwork (%) rmsd bond lengths (Å) bond angles (deg) no. of protein atoms no. of ligand atoms no. of solvent atomsb protein geometry preferred regions (%)c allowed regions (%)c outliers (%)c

space group cell dimensions a, b, c (Å) α, β, γ (deg) Rmerge (%) CC1/2 I/σ(I)a completeness (%)a redundancya

apo-SAV2435

Table 1. Crystallographic Data Collection and Refinement Statistics

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DOI: 10.1021/acs.biochem.6b00651 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry induced (OD600 = 0.5; 0.5 μM isopropyl β-D-1-thiogalactopyranoside), and grown for 4 h. These cells were harvested by centrifugation (4000 rpm, 20 min). The pellet was resuspended in lysis buffer [30 mM sodium phosphate (pH 7.8), 300 mM NaCl, 40 mM imidazole, 1 mM MgCl2, and 0.75 mM EDTA]. DTT (final concentration of 2 mM), protease inhibitor cocktail (final concentration of 100 μM), lysozyme (100 μg/mL), and PMSF (1 mM) were added and mixed for 30 min on ice. DNase I (final concentration of 1 μg/mL) was added and the mixture stirred for an additional 30 min. Cells were then lysed using a microfluidizer (110Y, Microfluidics International Corp.) and centrifuged at 15000 rpm for 15 min. The supernatant was collected and filtered with a 0.45 μm filter. The SAV2435 protein was then purified [binding buffer, which consisted of 30 mM potassium phosphate, 300 mM KCl, and 40 mM imidazole (pH 7.50); elution buffer, which consisted of 30 mM potassium phosphate, 300 mM KCl, and 300 mM imidazole (pH 7.50)] by HisTrap HP (GE Healthcare) chromatography using a linear gradient of elution buffer. SAV2435 was then concentrated and further purified by size exclusion chromatography [HiLoad 16/60 Superdex 75 (GE Healthcare)] on a size exclusion column in 30 mM Tris (pH 7.5), 100 mM NaCl, and 5% glycerol. Protein purity was found to be ∼99% from the Superdex 200 column by Coomassie Brilliant Blue-stained sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). C. tepidum genomic DNA was a generous gift from M. Madigan (Southern Illinois University, Carbondale, IL). The ct0179 gene was amplified from C. tepidum, subcloned into the pET28b+ (KanR) vector, and transformed into E. coli BL21(DE3) cells. The cells were grown, induced, harvested, resuspended, lysed, and clarified (centrifuged) as described for SAV2435. CTR107 was then purified by ion exchange (HiTrap Q HP, GE Healthcare) chromatography using a linear gradient. The binding buffer consisted of 30 mM Tris and 150 mM NaCl (pH 7.50), whereas the CTR elution buffer included 1.5 M NaCl. CTR was subsequently concentrated and further purified by gel filtration (HiLoad 16/60 Superdex 75, GE Healthcare) in 30 mM Tris (pH 7.5), 100 mM NaCl, and 5% glycerol. The protein purity was judged to be >95% by Coomassie Brilliant Blue-stained SDS−PAGE. Crystallization and Data Collection. Purified SAV2435 and CTR were concentrated to approximately 13 mg/mL (500 μM) in the gel filtration buffer. The protein concentrations were determined by UV−visible spectroscopy using the calculated ProtParam ε280 values (25900 M−1 cm−1 for SAV2435 and 16055 M−1 cm−1 for CTR107). For both SAV2435 and CTR107, crystals were grown via hanging-drop vapor diffusion at 18 °C by mixing equal volumes of a protein/ ligand solution (500 μM protein and 550 μM ligand) and a well solution [2.4 M ammonium sulfate and 0.1 M Tris (pH 8.5)]. Crystals were obtained in 4−7 days. Harvested crystals were stabilized and cryoprotected using a solution containing 2.4 M ammonium sulfate, 0.1 M Tris (pH 8.5), 30% glycerol, and the appropriate ligand(s) at 2 mM. The crystals were then flashfrozen in liquid nitrogen. All diffraction data were collected inhouse using a Rigaku FR-E SuperBright rotating anode X-ray generator (wavelength of 1.54 Å) and recorded with a Saturn 944+ CCD detector or an R-AXIS IV image plate detector. Data Reduction and Refinement. The data sets were indexed and scaled using HKL200043 and XDS.44 The crossvalidation, Rfree test sets (using 5% of the data) were generated, and space groups were confirmed using Aimless (CCP4).45 The

SAV2435 structures were refined against identical Rfree test sets using the SAV2435-TPP data as a reference. Phases were obtained by molecular replacement using Phaser (PHENIX).46 For SAV2435, all molecules of the asymmetric unit of the SAV2435 (JCSG) structure (PDB entry 3LUR) were used to generate a composite search model for ligand-free SAV2435 as well as the RH6G-, ET-, TPP-, and RH6G·TPP-bound structures. In the case of RH6G-bound CTR107, the ligandfree, NESG CTR107 structure (PDB entry 3E0H) was used as the initial search model. Early rounds of structural refinement included rigid-body, positional, and group B factor refinement as well as simulated annealing protocols, for all of which model building was performed in PHENIX and Coot. Later rounds included positional, individual B factor, and TLS refinements. The translation-libration-screw (TLS) parameters and groups were generated using the TLSMD server.47 The RH6G-bound CTR107 crystals were determined to be merohedrally twinned by XTRIAGE (PHENIX). As such, the appropriate twin law (h, −h − k, −l) was used in the refinement protocol described above. In the final stages of refinement, waters and other buffer and cryoprotectant-derived molecules were added to the models. The RH6G, ET, and TPP ligands were added to the models in early stages of the structural refinements. In all cases, the ligand-derived electron density was unambiguous and matched the shapes of the ligands. Additional refinements were assisted by simulated annealing omit and feature-enhanced maps (PHENIX). The quality and stereochemistry of the final structures were validated using Molprobity,48 which shows >97% of all residues in the most favored region of the Ramachandran plot; no residues were found in unfavorable conformations. Further assessment included real-space correlation B factor inspection. Crystallographic statistics are summarized in Table 1. Structural Analysis. Structural superimpositions and rmsd calculations were performed using Superpose (CCP4).49 HELANAL-Plus50 along with Mathematica 10.0 (Wolfram) was used to determine the helical geometrical parameters, including the helical axis vectors, helix center of mass−helix center of mass distances, and helical angles, which are defined as the angles between the helical axis vectors of the major α helices (αA and αB) in the GyrI-like domains. CASTp was used for the ligand pocket determinations as well as volume and surface area calculations.51 Accessible surface areas (ASAs) were calculated using NACCESS.52 Shape complementarity values were calculated using Sc (CCP4). 53,54 Ligand interactions were analyzed using the ligand−protein contact55 and plotted with LigPlot+.56 Bioinformatics. Structure-guided multiple-sequence alignments were performed using PROMALS3D using restraints derived from a previous sequence alignment as well as the coordinates of the GyrI-like domains.35,57 The resultant alignment and consensus sequence were visualized using Jalview.58 Binding Assays Performed by Intrinsic Fluorescence Quenching. Fluorescence spectra were recorded on a Fluorolog 3 (Horiba) fluorimeter at 25 °C. All samples as well as ligand (0.1−5 mM) and protein (10−100 μM) stocks were prepared in degassed, filtered fluorescence buffer [25 mM potassium phosphate (pH 7.4), 200 mM KCl, 1 mM EDTA, 2 mM DTT, and 5% spectrophotometric grade glycerol]. Frozen stocks contained 40% glycerol. The SAV2435, CTR107, and ligand concentrations of the working stocks were determined spectrophotometrically (see Crystallization and Data CollecD

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Biochemistry tion). For the ligands, molar absorptivity values from the literature were used. To monitor the intrinsic fluorescence in SAV2435, we used an excitation wavelength of 295 nm. Emission data were collected from 310 to 450 nm (λem,max = 340 nm). For CTR107, we used an excitation wavelength of 280 nm and collected emission data from 295 to 450 nm. The SAV2435 and CTR107 concentrations used for the KD determinations ranged from 200 to 500 nM. Gel filtration PD-10 columns (GE Healthcare) were used to exchange the proteins into the titration buffer system. All experiments included fluorescence and UV−vis measurements of working and reference samples. Ligands were added to the working and reference samples in a stepwise fashion (1−30 μL aliquots of 0.1−5 mM stocks in 2.5 mL of sample). A maximum of 0.2 mL of the ligand stock was added per experiment. Fluorescence and UV−vis spectra were recorded after a 1−2 min equilibration time. Corrected fluorescence intensities were obtained after dilution; background corrections and inner filter corrections for both the excitation and emission wavelengths were applied to obtain the final fluorescence values. All binding isotherms were generated using the corrected fluorescence intensities at 340 nm for SAV2435 and 310 nm for CTR107. The fluorescence-derived KD values were determined from numerical fits (Kaleidagraph, Synergy Software) to the data. The data were best described by a single-site binding model using hyperbolic (eq 1) or quadratic (eq 2) curve fits: ⎛ [L]T ⎞ Fcorr = Fmax − ΔF ⎜ ⎟ ⎝ KD + [L]T ⎠ Fcorr = Fmax ⎛ K + [L] + [P] − D T T − ΔF ⎜⎜ ⎝

Table 2. Helical Axis Angles, Distances between Helices, Pocket Volumes, and Pocket Surface Areas for GyrI-like Proteins protein

PDB entry

helical axis anglea (deg)

helix distanceb (Å)

pocket volumec (Å3)

pocket surface areac (Å2)

LIN2189 BmrR CTR107 GyrI Rob Cass2 SAV2435

3B49 3Q1M 3E0H 1JYH 1D5Y 3GK6 3LUR

78 78 89.5 108 125 133 141

33.3 32.2 30.1 25.3 23.8 22.2 19.5

2370 594 710 2067 628 1117 1518

1202 398 404 1016 401 645 736

a

The helical axis angle is defined as the angle between the helical axis vectors of the two major α-helices in the GyrI-like domain. The vectors were calculated using HELANAL-Plus, and the angle between the vectors was calculated in Mathematica 10.0.50 bThe helix distance is defined as the distance between the center of mass of the backbone atoms within each helix. cThe volume and surface area for the (putative) binding sites for GyrI-like proteins were calculated using CASTp.66 The molecular surface area was calculated for each pocket.67

SAV2435 structure was determined with three proteins in the asymmetric unit (Figure 2A), the newly determined ligand-free and ligand-bound structures show one molecule in the asymmetric unit (Figure 2B). Likewise, the CTR107 structure determined by NESG contains one molecule in the asymmetric unit (Figure 2C), while our CTR107−RH6G complex crystallized as a C2-symmetric dimer (Figure 2D). In all structures, including those containing multiple copies in the asymmetric unit, negligible structural differences are observed. For CTR107, the possibility of a dimeric structure in solution was discounted by AUC studies, which revealed monomeric species in the presence and absence of ligands at protein concentrations greater than 30 μM. The maximal pairwise rmsd observed for the SAV2435 Cα atoms is 0.514 Å. A value of 0.431 Å is obtained for the CTR107 proteins. SAV2435 and CTR107 present characteristics of the GyrIlike superfamily, despite sharing a low degree of sequence homology with each other and other GyrI-like family members (median pairwise identity of 10.1%; SD = 3.0) (Figure 3). Each single-domain protein displays a tandem duplication of SHS2 (β1−α−β2−β3) fold modules, which associate in an antiparallel fashion, yielding the GyrI superfamily (Figure 4).35 The SHS2 fold module shows a six-stranded antiparallel β-sheet core [(β2β4β3)A−(β2β4β3)B] and two α-helices (αA and αB) sitting on one face. The β1 strands are appendages to the SHS2 module that twist around to “cap” the β2 edges in a domainswapped manner (Figure 4A). The fold has been described as a “cupped hand” with the β-sheet forming the palm and α-helices αA and αB making up the thenar eminence and the fingers, respectively.34 The “cupped hand” shows pseudo-C2 symmetry with a groove between the α-helices. The helices are in an antiparallel arrangement and tilt relative to the strands of the βsheet core. In some cases, close helical approaches create an occlusion near the center of the groove, leaving two pseudosymmetry-related, concave surfaces with the potential to interact with small-molecule ligands. Pairwise structural superimpositions reveal a mean rmsd value of 3.50 Å (SD = 0.86), underscoring the overall similarities between the characterized GyrI superfamily members, with many of the differences being attributed to the placement of the major αhelices and loops (Figure 4C and Figure S1).

(1)

KD + [L]T + [P]T − 4[L]T [P]T ⎞ ⎟ ⎟ 2[P]T ⎠

(2)

where Fcorr is the corrected fluorescence, Fmax is the initial sample fluorescence, ΔF is the total fluorescence change over the course of the titration, [L] T is the total ligand concentration, [P]T is the total protein concentration, and KD is the ligand dissociation constant. The Fmax, ΔF, [P]T, and KD ligand values were determined from each curve fit. The affinity constants listed in Table 2 are averaged from at least two independent determinations.

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RESULTS A Structural Description of the GyrI-like Domains. The SAV2435 and CTR107 structures were determined by molecular replacement and refined using data collected to 1.73−2.10 Å. The data collection, reduction, and structure refinement statistics are listed in Table 1. The ligand-free SAV2435 structure (PDB entry 3LUR) determined by the JCSG and the ligand-free CTR107 structure determined by the NESG, residue side chains and solvent molecules excluded, were used as molecular replacement search models and starting models for the refinements. Initial rigid-body, positional, and overall B factor refinements, combined with simulated annealing, afforded well-defined, continuous protein electron density in all cases with the exception of several surface loops for which reasonable, albeit weak, electron density appeared during latter stages of the refinements. Space groups for SAV2435 and CTR107 structures reported here differ from those of structures previously determined by structural genomics consortia. Whereas the initial JCSG ligand-free E

DOI: 10.1021/acs.biochem.6b00651 Biochemistry XXXX, XXX, XXX−XXX

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Nearly all of the structures of the ligand-free single-domain GyrI proteins contain bound exogenous buffer and crystallization agents at the putative ligand-binding sites (Figure 5). The JCSG SAV2435 trimeric structure included PG4 and PGE. However, on the basis of the reanalysis of the ligand electron density in one of the protomers, we replaced the modeled PGE with another PG4 molecule (Figure 5A). The LIN2189 structure shows unmodeled electron density in its binding pocket that appears to be consistent with a HEPES buffer molecule (Figure 5B). The structure of Cass2 was modeled with 1PG; we have replaced 1PG with PE4 (Figure 5C). Like those of LIN2189, the coordinates of both NESG CTR107 and GyrI were deposited in the PDB with unmodeled electron density at their putative binding pockets. The data are consistent with PG4 and 12P molecules, respectively (panels D and E, respectively, of Figure 5). Importantly, a superposition of the remodeled structures indicates a common location for the GyrI superfamily ligand pocket. Despite the lack of overall sequence conservation, structural similarities appear to extend to the level of function. Similar to BmrR, the putative ligand-binding pockets of SAV2435, CTR107, LIN2189, Cass2, and GyrI are dominated largely by aromatic and, to a lesser extent, aliphatic residues. In all cases, both types together comprise approximately 70−80% of the binding pocket residues (Figure 6). One buried glutamate residue per structure in the ligand pocket (E135, E133, E185, E134, and E132, respectively) appears to be highly conserved among the GyrI-like domains. As observed in BmrR, these buried carboxylate side chains engage invariably in H-bonding contacts with nearby tyrosine side chains that likely participate in ligand recognition. These binding-site residues are highlighted in the structure-based sequence alignment shown in Figure 3. Finally, a comparison of the consensus sequence obtained from the alignment and that of BmrR reveals residues of similar hydrophobicity (aromatic and aliphatic) at seven of the eight positions occupied by the aromatic platform and hydrophobic pincer residues in the latter. Notable structural variations are observed for the different GyrI family members. Major differences involve the distances and angles between the two major helices, αA and αB (Figure S2). Not surprisingly, the loops also show highly variable structures. Incidentally, loop differences are even observed

Figure 2. Comparison among the multiple crystal forms and contents of the asymmetric units of SAV2435 and CTR107 in the presence and absence of ligand. (A) Structure of apo-SAV2435 (PDB entry 3LUR) determined by the JCSG. The asymmetric unit contains three monomers. Tetraethylene glycol (PG4) is depicted as spheres. (B) Structure of apo-SAV2435 reported here. The asymmetric unit contains a monomer. Glycerol (GOL) and bound waters are shown as spheres. (C) Structure of apo-CTR107 (PDB entry 3E0H) determined by the NESG. The asymmetric unit contains a single monomer. Triethylene glycol (PGE) is depicted as spheres. (D) Structure of the rhodamine 6G-CTR107 complex reported here. The asymmetric unit contains a C2-symmetric dimer. Shown in spheres are rhodamine 6G (RH6G) molecules bound to each monomer. (E) Structure of LIN2189 (PDB entry 3B49) with 4-(2-hydroxyethyl)-1piperazineethanesulfonate (HEPES) depicted as spheres.

Figure 3. Multiple-sequence alignment of SAV2435 and CTR107 with other GyrI-like proteins generated using Promals3B. GyrI-like proteins with known structure (GyrI, CTR107, BmrR, SAV2435, Rob, LIN2189, and Cass2) are shown in a multiple-sequence alignment. A consensus sequence is shown below, using the PROMALS3D consensus symbols. Residues in bold in the consensus sequence are >80% conserved. Contacts with aromatic residues are highlighted in purple, contacts with aliphatic residues green, contacts with polar residues cyan, and long-range electrostatic interactions red. F

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Figure 4. (A) Cartoon depiction of the topology of the GyrI-like domain. The helices and β-strands are depicted as cylinders and arrows, respectively. (B) Structure of the GyrI-like domain colored to match the cartoon. (C) Structural superimpositions of GyrI-like domains presented in Figure 3.

Figure 6. Schematic representation of ligand-binding contacts for GyrI-like domains. (A) SAV2435 (PDB entry 3LUR) modeled with tetraethylene glycol (PG4). (B) LIN2189 (PDB entry 3B49), modeled with 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES). (C) CTR107 (PDB entry 3E0H) modeled with triethylene glycol (PGE). (D) Cass2 (PDB entry 3GK6) modeled with tetraethylene glycol (PG4). (E) GyrI (PDB entry 1JYH) modeled with dodecaethylene glycol monomethyl ether (12P). Cutoff distances of 4.5 and 3.5 Å were used for hydrophobic interactions and hydrogen bonds, respectively. Residues are colored by type: green for hydrophobic residues, magenta for aromatic residues, red for acidic residues, and cyan for polar residues. Hydrogen bonds are colored black. This figure was generated using Ligplot+.

Figure 5. Modeling of electron density in the putative ligand-binding pockets for GyrI-like proteins deposited in the PDB. (A) SAV2435 (PDB entry 3LUR) modeled with tetraethylene glycol (PG4). (B) LIN2189 (PDB entry 3B49), modeled with a 4-(2-hydroxyethyl)-1piperazineethanesulfonate (HEPES) molecule. (C) CTR107 (PDB entry 3E0H) modeled with triethylene glycol (PGE). (D) Cass2 (PDB entry 3GK6) modeled with tetraethylene glycol (PG4). (E) GyrI (PDB entry 1JYH) modeled with 3,6,9,12,15,18,21,24,27,30,33undecaoxapentatriacontane-1,35-diol (12P). The electron density was modeled using Coot, and ligands are colored yellow. Maps are contoured at 1.0σ for 2Fo − Fc (blue mesh) and 2.0σ for Fo − Fc (green mesh).

and angles between the two helices (Figure 7A−G and Table 2). We observe a wide range of angles that vary from 78° (BmrR and LIN2189) to 142° (SAV2435). CTR107, GyrI, Rob, and Cass2 show values of 90°, 108°, 125°, and 133°, respectively (Table 3). Rather than emanating from changes in the β-sheet core, the helical angles are established by the length and conformation of the elements connecting the helices to the β-sheet core structure. Whereas one would expect a direct correlation between the interhelical angles and distance between helical arms, we observe an inverse relationship as calculated by the helical vector cross products (Figure 7H). This is due to the antiparallel helical arrangement. An

between the different ligand-bound SAV2435 structures. On the other hand, pairwise comparisons between the strands of the β-sheet core reveal a relatively constant structure across the examined protein set (see Figure S2). Importantly, the helical angle and distance differences observed for the GyrI proteins appear to influence the shapes and sizes of their ligand-binding pockets. To obtain descriptions of the helical arrangements for the structurally characterized GyrI-like domains, we used HELANAL-Plus to compute various parameters that characterize the geometry of α-helices in proteins, including helical axes, which are used to determine the distances (center to center) G

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Figure 7. Helical axis angle, distance between helices, pocket volume, and pocket surface area for GyrI-like proteins: (A) BmrR (78°, PDB entry 3Q1M), (B) LIN2189 (78°, PDB entry 3B49), (C) CTR107 (90°, PDB entry 3E0H), (D) GyrI (108°, PDB entry 1JYH), (E) Rob (125°, PDB entry 1D5Y), (F) Cass2 (133°, PDB entry 3GK6), and (G) SAV2435 (141°, PDB entry 3LUR). (H) There is an inverse relationship between helical distances and angles. (I) A larger angle between helices correlates with an increase in pocket volume. (J) Plot of helical center of mass distances vs pocket volume. The volume and surface area for the (putative) binding sites for GyrI-like proteins were calculated using CASTp.66 The molecular surface was calculated for each pocket.67 All surface areas are shown in square angstroms, and all volumes are shown in cubic angstroms.

Table 3. Ligand Recognition by GyrI-like Proteins ligand

a

RH6G

rhodamine 6G

ET

ethidium

TPP

tetraphenylphosphonium

protein

KD (μM)a

SAV2435 CTR BmrR SAV2435 CTR BmrR SAV2435 CTR BmrR Cass2

0.30 ± 0.01 0.78 ± 0.07 0.52 ± 0.10 0.99 ± 0.07 1.30 ± 0.44 1.90 ± 0.20 78.7 ± 10.2 15.6 ± 2.8 81.0 ± 5.6 0.20 ± 0.08

ΔG° (kcal/mol)a −8.89 −8.33 −8.56 −8.18 −8.02 −7.80 −5.60 −6.56 −5.58 −9.13

± ± ± ± ± ± ± ± ± ±

0.03 0.05 0.13 0.10 0.18 0.55 0.08 0.10 0.04 0.04

The values reflect two or three independent determinations. Reported errors for each KD value were propagated from multiple curve fits.

αB with additional residues from the loop. Interestingly, despite variable contributions, ligand-binding pockets, as a group, display similar groovelike structures. Structural Basis of Ligand Recognition by SAV2435. As seen for the JCSG apo SAV2435 structure, we observe electron density within the putative SAV2435 ligand-binding pocket when SAV2435 is crystallized in the absence of added small molecules (Figure 8A,B). We modeled this extra density as two glycerol molecules, which were introduced into the structure during cryoprotection. The presence of exogenous ligands and their interactions with SAV2435 highlight the functionality of the pocket, including the ability to interact with hydrophobic, polar, and charged ligand moieties. PG4 and one of the glycerol ligands dock in a hydrophobic wedge. The wedge is formed on one side by three residues from helix αA (I30, W34, and Y38) and a residue from the sheet core (Y71). The other side of the wedge is formed by two hydrophobic helix αB residues (P106 and V109) with additional residues from the loop preceding the helix (V105) and another loop connecting two strands of the core (T142). The bottom of the wedge is composed of Y137 and E135, the latter of which is

examination of the dependence of pocket size on helix−helix distance and angle suggests the possibility of correlated changes (Figure 7I,J); however, in both cases, GyrI and LIN2189 show larger than expected pocket volumes (Figure 7). Interestingly, significantly larger helical departures revealed by a pairwise alignment of LIN2189 and SAV2435 are consistent with the latter result (Figure S2). Additional crystal structures are required for a detailed analysis of the correlation between the geometric arrangement of the major helices and ligand pocket shape and size. Overall, both the helical angle and distance exert significant effects on the global shape and size of the GyrI protein ligandbinding pockets. Specifically, the helix−helix angle and distance dictate which elements within the GyrI domain structure contribute to the ligand pocket. For example, the ligand pocket for proteins with helix−helix angles of approximately 90° (LIN2189, BmrR, and CTR107) mostly contains residues from one helix (αA) on one pocket side with loop residues from the β-core dominating the other. In contrast, the pockets for proteins with helical angles greater than 108° (Rob, Cass2, and SAV2435) exhibit major contributions from residues in αA and H

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Figure 8. Structural basis of ligand binding to SAV2435. (A) Apo-SAV2435 structure, in which the extra electron density is modeled with two glycerol (GOL) molecules. (B) SAV2435 modeled with tetraethylene glycol (PG4) (PDB entry 3LUR). (C) Binding of rhodamine 6G (RH6G) to SAV2435. (D) Binding of ethidium (ET) to SAV2345. (E) Alternate binding of tetraphenylphosphonium (TPP) to SAV2435. (F) Complex of tetraphenylphosphonium (TPP) and rhodamine 6G (RH6G) bound to SAV2435. The carbons for the residues of SAV2435 are shown as sticks for hydrophobic residues (green), aromatic residues (pink), and polar residues (cyan). The carbons for ligands are colored as blue sticks. Below the structures are 2Fo − Fc omit electron density maps (blue mesh) contoured at 1.0σ for rhodamine 6G (RH6G). The hydrogen bonding distances have been omitted for the sake of clarity. The distances range from 2.5 to 3.4 Å.

wedge core involve the hydroxyl moieties of Y38, Y138, and, possibly, Y71. Importantly, the interactions in the ligand-free SAV2435 structures match those observed in the RH6G- and ET-bound SAV2435 complexes (Figure 8C,D and Figure S3A,B). For both structures, we observed well-defined electron density within the wedge, which facilitated unambiguous modeling. RH6G and ET bound within the same groove as the cyroprotectant molecules observed in the apo structure, and both ligands interacted largely with aromatic and aliphatic residues lining the wedge. Specifically, the xanthene ring of RH6G engages in aromatic (π−π) stacking contacts with W34

highly conserved among the GyrI-like domains. Interestingly, the wedge is “capped” on both ends by flexible, polar residues. On one end are two glutamine residues (Q27 and Q110 from α A and α B , respectively) that adopt variable rotamer conformations in the different SAV2435 structures. The other end also contains a glutamine (Q47) and an acidic residue (D139); these too appear to be able to adopt variable conformations. Overall, these structures present a pocket displaying a largely hydrophobic core and a polar outer perimeter that interacts with the exogenously bound ligands and pocket-bound water molecules via hydrogen-bonding contacts. Hydrogen-bonding contacts observed within the I

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Biochemistry that are stabilized by hydrophobic contacts formed on the opposite side of the wedge pocket. Additional van der Waals contacts involve both RH6G ethyl amino substituents as well as I30, V109, and T142. For ET, the dominant interaction includes hydrophobic contacts between the ET phenanthridine ring and residue W34, the V105P106 pair, and minor contributions from T142. On the bottom of the wedge, RH6G and ET engage via edge-to-face contacts with Y137 and apparently, long-range electrostatics with E135. Both ligands also exhibit analogous polar interactions, including hydrogen bonds with Y39. RH6G engages directly with Q27 via a hydrogen bond, where ET interacts with the same residue via a water-mediated contact. An Alternate SAV2435-Binding Pocket. In contrast to that with ET and RH6G, the structure of SAV2435 bound to TPP contained electron density in a different pocket within the GyrI domain (Figure 8E and Figure S3C). The location of the density is proximal to the two helices but directly opposite from that of the canonical binding site occupied by RH6G, ET, and glycerol. Given the pseudo-C2 symmetry of the GyrI domain structure, the observed binding of TPP in this region is not completely surprising. However, it is unique among all of the GyrI X-ray structures determined to date. Unlike the putative binding site, the newly identified pocket displays a bowl shape and appears to be more hydrophobic than the region accommodating RH6G and ET. The rim of the bowl is composed of three helix αB residues, M113, H117, and H121. The remaining rim-defining residues are contributed by a single loop (αB−β2). These include hydrophobic contributions by T127, K129, and F133. Two aliphatic residues, I120 and P154, sit on the bottom of the bowl-like pocket. In this binding mode, three of the four phenyl substituents of TPP interact with the rim of the alternative pocket; the remaining phenyl group interacts with I120, T127, F133, and P154 and is completely removed from solvent. Finally, a number of solvent molecules are observed proximal to the bound TPP. Although they do not engage directly with TPP, they do interact with elements forming the ligand-binding pocket. Ternary, Two-Ligand-Bound SAV2435 Complex. To establish mutually exclusive binding of ligands at the putative and alternative pockets, we crystallized SAV2435 in the presence of both RH6G and TPP (Figure 8F and Figure S3D). The ternary complex displays negligible differences compared to the singly bound RH6G- and TPP-bound complexes with rmsd values of 0.154 and 0.222 Å, respectively. As the ternary complex presents protein−ligand contacts that are nearly identical to those of both singly bound adducts, a ligand bound at one site does not appear to influence the conformation of the other site. Moreover, whereas superimpositions of the SAV2435 structures reveal a rather rigid putative ligand-binding pocket, the same analyses suggest a slightly more flexible TPP-binding pocket. Small differences result from rotameric variations at four positions, namely, H117, H120, N124, and K129. The largest changes are observed for H117 and H120, both of which adopt conformations that open the pocket for TPP binding. Ligand-Free and Ligand-Bound CTR. The ligand-free NESG CTR107 structure also contains the exogenous cryoprotectant molecule, PG4, bound at the putative ligandbinding pocket (Figure 9A). This ligand is not present in the original deposited X-ray structure; however, analysis of the data reveals a significant amount of unmodeled electron density in the region in which ligands are expected to bind. Like that of

Figure 9. Structural basis of ligand binding to CTR107. (A) Binding of triethylene glycol (PGE) to CTR107 (PDB entry 3E0H). (B) Binding of rhodamine 6G to CTR107. Rhodamine 6G (RH6G) is shown as blue sticks. Shown below panel B is the 2Fo − Fc omit electron density map (blue mesh) contoured at 1.0σ for rhodamine 6G (RH6G).

SAV2435, binding of PG4 to CTR107 reveals a hydrophobic wedge-shaped pocket with one side being formed by three helix αA residues (F35, Y39, and L43) as well as Y75, which is derived from the sheet core; the other side is formed by two loop residues (N138 and P139). The bottom of the wedge includes an aromatic (Y135) residue along with P56 and P57. The wedge is effectively capped at one end by two aromatic (Y61 and Y106) residues and the highly conserved E133, all of which are positioned to interact with ligands via hydrogenbonding contacts and electrostatic interactions, respectively. In contrast to SAV2435, the putative CTR107 pocket contains only two polar residues, leaving it with a more limited capacity to engage in hydrogen-bonding contacts. However, as seen in SAV2435, internal tyrosine residues appear poised to participate in polar interactions. The RH6G-bound CTR107 complex recapitulates key features observed for PG4 binding (Figure 9B and Figure S3E). As with SAV2435, there is well-defined electron density for the ligand. In contrast to the SAV2435 structure, the RH6G ligand adopts a slanted orientation in wedge, which prevents steric clashes with P57. As observed with the SAV2435 analogue, the RH6G xanthene ring rests against several aromatic residues lining one side of the wedge with “clamplike” stabilizing contributions from the other side. Interestingly, we observe two conformations for Y106. One is the capping conformation observed in the PG4-bound structure. The other features a rotation about the Cα−Cβ bond that moves the Y106 aromatic ring to the side of the wedge. Both rotamers appear to be equally populated. No polar interactions are observed for the ethyl amino substituents, both of which expose the nitrogen atoms to solvent. The lone hydrogen-bonding contact links the carbonyl oxygen of the ethyl benzoate moiety to N138. Solution Analyses of Ligand Recognition by SAV2435. To further evaluate ligand recognition by SAV2435 and CTR107, we examined their solution binding properties with a number of ligands, including RH6G, ET, and TPP. To do so, J

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Biochemistry we exploited the presence of prominent aromatic residues in the ligand-binding pockets of both proteins. SAV2435 contains two tryptophan residues, one of which appears to play a major role in the binding of ligands. In contrast, the CTR107 structures reveal tyrosine residues as major determinants of ligand binding. As such, we monitored ligand-dependent changes in the intrinsic tryptophan fluorescence for the former and tyrosine fluorescence for the latter. Both show saturable, ligand- and concentration-dependent decreases in their intrinsic fluorescence (Figure 10). The SAV2435 tryptophan fluores-

Whereas the biological activities of isolated GyrI-like domains remain largely unknown, a high percentage (41%) among known sequences suggests an important biological function. Our structural data support previous bioinformatics analyses of GyrI-like domains. Moreover, the predicted global features of the GyrI-like domain are recapitulated by our structural data. Aravind et al. described a putative ligand-binding site at the interface of the two tandem copies of SHS2 modules that form GyrI-like domains.35 We observe ligand-derived electron density in this precise region for all structurally characterized GyrI-like proteins presented in this study and determined by structural genomics consortia JCSG and NESG. Crystallographic and solution binding analyses of Cass2 and BmrR provide additional examples of such binding modes.23,40 Whereas GyrI, the founding member of the GyrI-like superfamily, has been implicated as an inhibitor of DNA gyrase, the presence of electron density in its putative binding pocket suggests a potential alternative ligand binding activity for this system.41 On the basis of sequence conservation, Aravind et al. also predicted a set of ligand-contacting residues.35 These consist largely of hydrophobic residues within the β-core. For BmrR and GyrI, many of the predicted residues have been shown to participate in ligand binding or play key roles in stabilizing the core structure.23,41 This prediction highlights a glutamate residue located in the middle of the core that offers electrostatic stabilization for the recognition of cationic ligands. Importantly, binding contributions from both helices (αA and αB) were not identified, likely because of the lack of sequence conservation in these regions. For the single-domain GyrI proteins characterized to date,37,40,41 we observe similar groovelike, ligand-binding pockets that are dominated by aromatic and aliphatic residues as well as a buried glutamate residue that may mediate the recognition of lipophilic cations. Several biochemical and structural investigations have linked multiple GyrI-like proteins to MDR-related functions. BmrR provided the first example of MD binding by a GyrI-like domain.27,32 Subsequently, solution NMR studies of Rob, which contains a C-terminal GyrI-like domain, also reveal the ability to bind structurally diverse ligands.37−39 Structural, biochemical, and genetic analyses indicate a similar function for Cass2.40 Although GyrI appears to be an exception, as stated above, the structure of its binding pocket is compatible with ligand binding.41,59,61 Whereas the binding properties of LIN2189 have yet to be determined, identification of a small molecule bound to its putative ligand-binding pocket suggests a similar function (PDB entry 3B49). Finally, our solution and Xray analyses of SAV2435 and CTR107 highlight the MD binding function for the GyrI-like domain, consistent with the observations for Cass2 and BmrR. Although more crystallographic depictions of binding are needed to fully elucidate determinants of MD recognition by SAV2435 and CTR107, a number of elements are apparent from the structures obtained to date. Interestingly, neither SAV2345 nor CTR107 presents a pseudosymmetric aromatic ligand docking pad similar to that observed in BmrR.23 However, the aromatic-rich, groovelike structures of both SAV2435 and CTR107 appear to perform a similar function (Figures 8A−F and 9A,B), which is conducive to the docking of unrelated ligands. Moreover, BmrR,23,32,33 SAV2435, and CTR107 employ similar “clamplike” motifs that further stabilize bound ligand. The SAV2435 and CTR107 pockets also utilize a

Figure 10. Drug binding quenches the intrinsic fluorescence of SAV2435 and CTR107. (A) The intrinsic tryptophan fluorescence (λex = 295 nm; λem = 340 nm) of SAV2435 (pink circles) is plotted as a function of increasing rhodamine 6G concentration. (B) The intrinsic tyrosine fluorescence (λex = 280 nm; λem = 310 nm) of CTR (cyan squares) is plotted as a function of increasing rhodamine 6G (RH6G) concentration. Each titration was fit to eq 1 via nonlinear regression. Numerical fits to the binding isotherm data for all ligands are consistent with the identical, independent-site model. Insets show emission spectra of SAV2435 (λex = 295 nm) and CTR (λex = 280 nm) with increasing concentrations of rhodamine 6G. Arrows show the decrease in fluorescence intensity upon ligand titration.

cence is almost completely quenched by some ligands, suggesting that the other tryptophan residue (W152) does not contribute significantly. Given that the W152 side chain is hydrogen-bonded to the carboxylate moiety of E150, this is not surprising. Moreover, the fluorescence changes observed for CTR107 indicate selective quenching of the tyrosine contributions. The data are summarized in Figure 10. The resultant data are described well by the simple, singlesite binding model. The KD values determined by the curve fits are listed in Table 3. Interestingly, despite notable differences in pocket structures, the ligand affinities observed for SAV2435 and CTR107 mirror those observed for BmrR, a known MDR protein.59 Among the ligands tested in this study, RH6G exhibits the highest affinity for both proteins (0.23 and 0.78 μM, respectively) followed by ET (0.99 and 1.30 μM, respectively). In both cases, we observe similar affinities for BmrR (0.52 μM for RH6G and 1.90 μM for ET).30 For SAV2435, CTR107, and BmrR, TPP, which is a widely used MDR probe, shows the weakest binding with affinities of 78.7, 15.6, and 81.0 μM, respectively. In both structurally characterized TPP-bound complexes (BmrR and SAV2435), TPP adopts alternative binding modes.

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DISCUSSION Analysis using the PFAM server reveals that GyrI-like domains are widespread among bacteria, eukaryotes, and archaea.35 Although examples of stand-alone GyrI-like domains are common, many are found fused to other protein domains, such as DNA-binding domains or enzymatic motifs.23,35,36,60 K

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recognition, as both proteins bind to MDR probes with affinities matching those of known MDR regulators. These results, along with previous bioinformatics analysis of GyrI-like domains, suggest an evolutionarily conserved function for GyrIlike domains related to small-molecule binding and MDR.

number of hydrogen-bonding donor and acceptor sites to accommodate ligands displaying diverse polar properties. SAV2434 and CTR107 share key ligand binding characteristics with other MDR proteins, including BmrR, QacR, TtgR, and CmeR. First, the ligand accessible surface area (ASA) burial values range between 70 and 80% for SAV2345 and CTR107. Such values are lower than those observed for highly specific systems, where ligand burial values are often >80%. Second, SAV2435 and CTR107 bind ligands with dissociation constants in the high nanomolar to low micromolar range, which typify affinity measurements for the other MDR proteins. On the other hand, SAV2435 and CTR107 depart from canonical models of MDR recognition in terms of ligand−receptor shape complementarity. Shape complementarity (Sc) values for SAV2435 (0.75) and CTR107 (0.79) are significantly higher than for other MDR proteins such as BmrR (0.57), QacR (0.65), TtgR (0.64), and CmeR (0.59). Interestingly, the values are on par or just slightly less than those observed for systems that display highly discriminatory properties in ligand binding. Whether this high level of shape complementarity is a general feature of GyrI-like ligand recognition remains to be determined, as the higher values observed for SAV2435 and CTR107 may reflect a structural bias or be due to the small sample size with respect to ligand selection. It is not surprising that close relatives of GyrI-like proteins are found fused to DNA-binding motifs, enzymes, and other effector domains.35 However, placing what appears to be a simple ligand-binding function in the context of bacterial, archaeal, or even eukaryotic cellular physiology is a challenge. To this end, the cellular functions of SAV2435, CTR107, LIN2189, and other single-domain GyrI proteins remain unexplored and unknown. However, the genomic contexts of a number of GyrI proteins are consistent with potential ligand binding and resistance functions. Indeed, analyses of the neighboring genes of those encoding the SAV2435, CTR107, LIN2189, and GyrI proteins, in each case, reveal genes annotated as putative small-molecule transporters (Figure S4). A number of functions can be envisioned for the GyrI proteins. One possible function is drug sequestration. Previous studies have shown that the bleomycin resistance proteins and proteins such as TipAS are expressed to sequester specific antibiotics.62−64 It is appealing to hypothesize that singledomain GyrI proteins perform analogous functions by binding to harmful xenobiotic agents. Another possibility is that GyrIlike proteins could dock onto MarA-like or other DNA-binding domains to form two-element transcription factors.40 For SAV2435, the noncanonical TPP-binding site may function as a protein-binding interface to form a two-component drugsensing system. Interestingly, the residues lining the SAV2435 TPP-binding pocket are highly conserved (Figure S5). Unlike eukaryotic systems, two-element transcription factors in bacteria are uncommon.65 As such, ligand-responsive association in bacterial transcription would be novel and potentially provide a means for combinatorial regulation. In summary, with the exception of TPP, we observe that ligands bind to a conserved aromatic-rich groove of the GyrIlike domain proteins SAV2435 and CTR107. In addition, analysis of structures determined by structural genomics consortia reveals unmodeled electron density at the same location. TPP adopts an alternate binding mode opposite the canonical binding site, which may reflect an interface for protein−protein interactions. Solution binding studies are consistent with a role for SAV2435 and CTR107 in xenobiotic

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.6b00651. Pairwise structural comparisons of GyrI proteins against SAV2435 (PDB entry 3LUR) (Figure S1), rmsd values (in angstroms) derived from pairwise superimpositions of the GyrI proteins (Figure S2), schematic representation of ligand-binding contacts to SAV2435 and CTR107 (Figure S3), genomic contexts of (A) E. coli GyrI (sbmC), (B) C. tepidum Ctr107 (ct0179), (C) L. innocua LIN2189, and (D) S. aureus SAV2434 that reveal potential neighboring ligand transporter and other resistance functions (Figure S4), and multiple-sequence alignment of homologues (