Structural Insights into Substrate Recognition by Clostridium difficile Sortase

Sortases function as cysteine transpeptidases that catalyze the covalent attachment of virulence-associated surface proteins into the cell wall peptidoglycan in Gram-positive bacteria. The substrate proteins targeted by sortase enzymes have a cell wall sorting signal (CWSS) located at the C-terminus. Up to date, it is still not well understood how sortases with structural resemblance among different classes and diverse species of bacteria achieve substrate specificity. In this study, we focus on elucidating the molecular basis for specific recognition of peptide substrate PPKTG by Clostridium difficile sortase B (Cd-SrtB). Combining structural studies, biochemical assays and molecular dynamics simulations, we have constructed a computational model of Cd-SrtBΔN26–PPKTG complex and have validated the model by site-directed mutagensis studies and fluorescence resonance energy transfer (FRET)-based assay. Furthermore, we have revealed that the fourth amino acid in the N-terminal direction from cleavage site of PPKTG forms specific interaction with Cd-SrtB and plays an essential role in configuring the peptide to allow more efficient substrate-specific cleavage by Cd-SrtB.


INTRODUCTION
Bacterial surface proteins are crucial virulence factors that mediate adhesion to the host as the first step establishing an infection. The sortase family of cysteine transpeptidases catalyzes the anchoring of a wide variety of virulence-associated surface proteins to the cell wall peptidoglycan (Spirig et al., 2011;Cascioferro et al., 2014;Bradshaw et al., 2015). Sortases, unique to Gram-positive bacteria, recognize and cleave the C-terminal cell wall sorting signal motif (CWSS) of substrate proteins (Schneewind et al., 1992(Schneewind et al., , 1993Paterson and Mitchell, 2004). Based on the primary sequences and their roles in biological functions, sortases are classified into six classes: A, B, C, D, E, and F. Class A sortases (SrtAs) are present in almost all Gram-positive bacteria. The first identified and best-known class A enzyme is the Staphylococcus aureus SrtA (Sa-SrtA), which anchors at least 19 LPXTG-containing surface proteins Perry et al., 2002;Spirig et al., 2011;Bradshaw et al., 2015). Sa-SrtA mutants exhibited a severe reduced adherence to epithelial cells and virulence in animal models (Flock et al., 1987;Mazmanian et al., 2000;Clancy et al., 2010). SrtAs are commonly called housekeeping sortases, whereas the remaining five classes are the accessory sortases. Class B sortases (SrtBs) recognize the NXXTN motif rather than the classical LPXTG motif and have distinct functions (Comfort and Clubb, 2004;Dramsi et al., 2005); some members of this group are involved in iron acquisition, whereas sortase B of Streptococcus pyogenes is involved in pili assembly (Kang et al., 2011). Class C sortases (SrtCs) are essential for pili polymerization in many species (Huang et al., 2010), such as Enterococcus faecalis (Kline et al., 2009), Corynebacterium diphtheria (Ton-That and Schneewind, 2003;Gaspar and Ton-That, 2006), Streptococcus agalactiae (Dramsi et al., 2006;Cozzi et al., 2012), and Streptococcus pneumonia (Fälker et al., 2008;LeMieux et al., 2008;Manzano et al., 2008). In addition, SrtC is required for aerial hyphae formation in Streptomyces coelicolor (Di Berardo et al., 2008). Class D sortases (SrtDs) are similar to SrtAs and perform a housekeeping role; they most frequently present in Bacillus species and are involved in spore formation (Marraffini and Schneewind, 2006). Recent studies have reported that Clostridium perfringens SrtD is structurally and catalytically distinct from Bacillus anthracis SrtD, suggesting that C. perfringens SrtD may display a different aspect of the SrtD family (Marraffini and Schneewind, 2006;Suryadinata et al., 2015). Class E and F sortases are mainly identified in Actinobacteria; they share a limited primary sequence homology with other sortases and their functions remain undetermined (Comfort and Clubb, 2004;Dramsi et al., 2005;Spirig et al., 2011).
In the genome of toxigenic C. difficile strain 630, only one functional sortase, the SrtB gene, is present (Donahue et al., 2014). C. difficile is a Gram-positive, anaerobic, and sporeforming bacterium that can colonize the gut if the normal intestinal microbiota is disrupted (Kelly and LaMont, 1998). C. difficile infection (CDI) is highly associated with antibiotic therapy and has been recognized as the leading cause of antibiotic-associated diarrhea, making it a major public health threat worldwide (Henrich et al., 2009;Bagdasarian et al., 2015). In the United States alone, CDI causes approximately 15,000-20,000 deaths annually, and CDI-associated hospitalizations among the general population doubled from 31 to 61 per 100,000 from 2008 to 2010 (Viseur et al., 2011). Furthermore, the CDI risk is high in patients receiving antibiotic treatments because their gastrointestinal flora is unfavorably altered. CDI manifestations can include asymptomatic colonization, mild to severe chronic diarrhea, pseudomembranous colitis, and death because of multiple organ failure (Kelly and LaMont, 2008). At present, metronidazole and vancomycin are mainly administered for treating CDI. However, up to 25% of patients treated for CDI experience recurrences after discontinuing antibiotic therapy (Bartlett et al., 1980;Tedesco et al., 1985;Leffler and Lamont, 2009;Surawicz et al., 2013). The increase in treatment failure or multiple relapses have raised a concern. An alternative therapy, fecal microbiota transplantation, has been used to restore healthy gut flora in patients with recurrent CDI (Rohlke and Stollman, 2012;Dodin and Katz, 2014;Duke and Fardy, 2014). Fecal transplantation is highly effective; however, it is still not widely accepted. In the last decade, sortase has been identified as a promising anti-infective therapeutic target (Zong et al., 2004b;Maresso et al., 2007;Suree et al., 2009;Oh et al., 2010;Jacobitz et al., 2014;Zhang et al., 2014), thus offering an encouraging avenue toward the development of drugs against CDI.
There have been many structural and functional studies on sortases from various Gram-positive pathogens (Spirig et al., 2011;Cascioferro et al., 2014;Bradshaw et al., 2015), and studies on C. difficile sortases were reported recently (Donahue et al., 2014;van Leeuwen et al., 2014;Chambers et al., 2015). Sa-SrtA has been extensively studied, and the catalytic mechanism underlying how Sa-SrtA anchors the surface protein to cell wall has been reported Perry et al., 2002). The membrane-bound Sa-SrtA scans and recognizes the LPXTG sequence of the CWSS, and a nucleophilic attack from the active thiol group of sortase cysteine residue to the peptide bond between the threonine and glycine of the LPXTG motif results in the formation of a thioester intermediate Ton-That et al., 1999;Perry et al., 2002). The sortaseacyl intermediate is then resolved by the nucleophilic attack of a free amino group within lipid II, resulting in the release of the surface protein from the sortase onto the cross bridge of the newly formed peptidoglycan (Frankel et al., 2005). This substrate release restores the enzyme active site, allowing the sortase to process more substrates (Frankel et al., 2005).
Recent studies have demonstrated that C. difficile SrtB (Cd-SrtB) can recognize and cleave (S/P)PXTG between threonine and glycine; however, Cd-SrtB cannot recognize the sequence LPXTG and NPQTN, corresponding to the recognition motifs for Sa-SrtA and Sa-SrtB, respectively (van Leeuwen et al., 2014;Chambers et al., 2015). It remains unclear how structurally similar sortases achieve substrate specificity. Thus far, the available structures of sortase-substrate complexes are limited to the nuclear magnetic resonance structure of Sa-SrtA bound to an LPATG substrate analog (Suree et al., 2009), a crystal structure of a Sa-SrtA mutant complexed with LPETG (Zong et al., 2004a), and a crystal structure of Sa-SrtB covalently bound to an NPQTN analog (Jacobitz et al., 2014). Therefore, studying and comparing new structures of sortases and sortase-substrate complexes from a wide range of organisms will enhance our understanding of how sortases recognize their respective substrates. In this study, we determined the crystal structure of the catalytically active SrtB from C. difficile and constructed a probable model of the Cd-SrtB-PPKTG complex by computer modeling and molecular dynamics simulations to gain structural insights into the substrate specificity for Cd-SrtB.

Protein Overexpression and Purification
The SrtB N26 from C. difficile 630 was cloned into a pMCSG7 vector by using a ligation-independent cloning method (Aslanidis and de Jong, 1990) and transformed into E. coli BL21 (DE3). A recombinant 6xHis-tagged SrtB N26 protein was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside when the cells reached an O.D. 600 of 0.5, and further incubated at 37 • C for 4 h. Cells were centrifuged at 8000 rpm for 30 min at 4 • C, resuspended in buffer A (20 mM HEPES pH 7.4, 200 mM NaCl and 20 mM imidazole), and disrupted by sonication on ice. Moreover, the supernatant was loaded into an Ni-NTA column (GE Healthcare Life Sciences) and contaminant proteins were eliminated through a washing procedure by using 60 mM imidazole in buffer A. SrtB N26 proteins were eluted with 300 mM imidazole in buffer A. Fractions containing SrtB N26 proteins were pooled and further purified through HiLoad 26/600 Superdex TM 75 size-exclusion chromatography (GE Healthcare Life Sciences). Subsequently, the proteins were dialyzed in buffer B (10 mM HEPES pH 7.4 and 150 mM NaCl) and stored at 4 • C for further use.

FRET-Based Assay
The peptide substrate of Cd-SrtB N26 , PPKTG was conjugated using a fluorophore, 5-[(2-aminoethyl) amino] naphthalene-1sulfonic acid, and a quencher, 4-([4-(dimethylamino) phenyl] azo) benzoic acid. To determine the suitable concentrations of Cd-SrtB N26 and fluorescently labeled PPKTG peptide in the assay, a matrix of various enzyme and substrate concentrations in the total volume of 100 µL in FRET buffer (10 mM HEPES pH 7.4 and 150 mM NaCl) was reacted in a 96-well black polystyrene plate and was incubated at 37 • C for 48 h. The fluorescence signal was monitored at an excitation/emission wavelength of 340/490 nm and recorded every hour during the first 8 h and then at 24, 36, and 48 h by using a Spectra-Max M3 plate reader (Molecular Devices). The optimal concentrations of Cd-SrtB N26 and fluorogenic peptide used in our reactions are 240 and 20 µM, respectively. Stock solutions of MTSET and AAEK1 were dissolved in the FRET buffer, and curcumin was dissolved in DMSO. Serial dilutions of inhibitors at the millimolar range were added into the FRET buffer. All experiments were conducted in triplicate. The data are presented as means and standard errors. The statistical significance of the inhibitory effect on enzymatic activity was calculated using GraphPad Prism software (GraphPad Software). Twotailed unpaired Student t-tests revealed significant differences between Cd-SrtB N26 + PPKTG and Cd-SrtB N26 + PPKTG + inhibitors at different concentrations ( * p ≤ 0.05, * * p ≤ 0.01, and * * * p ≤ 0.001).

Crystallization
Purified Cd-SrtB N26 proteins were concentrated to 8-10 mg/mL for crystallization trials. For sparse matrix screening, numerous commercial kits (Hampton Research and Emeralds BioSystems) were used for performing the crystallization setup of the vapor diffusion method by using a high-throughput platform (Digilab Genomic Solutions). Cd-SrtB N26 crystals were observed in sitting drops containing 0.5 µL of protein and 0.5 µL of various crystallization solutions at 25 • C within 1 week. Diffraction quality crystals were obtained using the hanging drop method by mixing 1 µL of protein (10 mg/ml in 10 mM HEPES pH 7.4 and 150 mM NaCl) and 1 µL of solution (0.1 M citric acid pH 3.5, 24% PEG 3350 and 0.1 M glycine). Prior to data collection, the crystals were directly mounted on loops from mother liquor and flashfrozen in liquid nitrogen without an additional cryoprotectant treatment.

X-Ray Data Collection and Processing
Diffraction data were collected on beamline BL13B1 of the National Synchrotron Radiation Research Center (NSRRC; Hsinchu, Taiwan) and beamline 4.2.2 of the Advanced Light Source (Berkeley, CA, USA). Most of our sortase crystals did not diffract beyond 3 Å resolution. The best crystal diffracted to 2.67 Å resolution and native data were collected at BL13B1 of the NSRRC. Ninety frames were collected, each with 1 • oscillation and were exposed for 30 s at the wavelength of 1.0 Å with the crystal-to-detector distance of 400 mm at a temperature of 100 K. The data were indexed, integrated, and scaled using HKL2000 (Otwinowski and Minor, 1997). The initial data were scaled to 2.67 Å resolution, but the I/σI decreased to 1.67 at the highest resolution shell (2.77−2.67 Å) suggesting that the data were effective at a resolution of approximately 2.8 Å. The crystallographic parameters and data collection statistics were summarized in Table 1.

Structure Determination and Refinement
The crystal structure of Cd-SrtB N26 was solved by molecular replacement method by program Phaser-MR (Mccoy et al., 2007) with the structure of Sa-SrtB (PDB 1QWZ) (Zong et al., 2004a) as a search model. Initially, the structure was determined at 3.5 Å resolution, and a polyalanine model was constructed. With the availability of better native datasets at higher resolutions, the model was manually rebuilt using COOT (Emsley et al., 2010) guided by 2Fo-Fc and Fo-Fc density maps. Computational refinement was conducted using REFMAC (Murshudov et al., 2011) and PHENIX (Adams et al., 2010), with 5% of the data flagged for cross-validation. We first carried out the refinement at 2.67 Å resolution, but the statistics were poor. The structural quality was improved when we systematically excluded the weak inflections by truncating data at different resolutions. Iterative model rebuilding and refinement were conducted. The final refinement statistics for the structural model at 2.8 Å resolution were summarized in Table 1. Coordinates and structure factors with the identifier 5GYJ have been deposited in the Protein Data Bank.

MD Simulations
The peptide of sequence NPQC co-crystalized with S. aureus SrtB structure (PDB 4LFD) was positioned in the catalytic pocket of C. difficile SrtB by superimposing of S. aureus SrtB onto C. difficile SrtB (Cd-SrtB). It was then mutated into a set of peptides of our interest, including PPKT and NPQT. PPKTG and NPQTN were modeled by adding one more G and N, respectively, in the C-terminus using the package VMD 1.9.2 (Humphrey et al., 1996). We further replaced the P4 residue in PPKTG to give SPKTG and NPKTG. Missing loops of 27-28 (ML), 162-167 (ESDYDY), 210-216 (TYEFDDA), and 225 (I) in the Cd-SrtB were modeled by UCSF CHIMERA (Yang et al., 2012). Cd-SrtB−peptide complexes were solvated in TIP3P water molecules of 8Å thickness in all directions of  a rectangular box (Jorgensen et al., 1983). One hundred and fifty millimolar sodium chloride were added as counter-ions to neutralize the system. Energy minimization and explicitsolvent MD simulations were performed by NAMD 2.10 package (Phillips et al., 2005;Huang and MacKerell, 2013) with CHARMM36 force field (Huang and MacKerell, 2013). Nonbonded interactions were carried out using a cut-off distance of 12 Å, with a switching distance of 10 Å. With periodic boundary conditions, the Particle Mesh Ewald method was employed for calculations of electrostatic energy (Darden et al., 1993). The Cd-SrtB, crystal waters and peptides (PPKTG, SPKTG, NPKTG, and NPQTN) were first restrained to the positions reported in the X-ray crystallography and then gradually released, first on the side chains and then the entire peptide and protein. No hydrogen atom is restrained at all time. After a 1.25 ns canonical ensemble (NVT) heating process and a short isothermalisobaric (NPT) equilibration, the whole system was allowed for a productive run for 9 ns in a NPT ensemble at 310K and 1 atm, respectively controlled by solvent friction and Nosé-Hoover Langevin piston (Feller et al., 1995). MD simulations trajectories are further analyzed by VMD, MDAnalysis toolkit (Michaud-Agrawal et al., 2011) and in-house programs coded in python.

Contact Frequency Analysis for the P4 Residue of Different Substrate Peptides
To understand the role of the P4 residue in the substrate peptides, we analyzed the contact frequency of the P4 residue of a peptide with Cd-SrtB N26 . At every frame, a residue in Cd-SrtB N26 situating within 4.0 Å from the P4 residue of the substrate peptides is marked as a contact. For each contacting residue, the contacting percentage is defined as the number of frames that the residue is in contact divided by total number of frames in MD simulations.

Root-Mean Square Fluctuations (RMSF) Analysis for Peptide Residues
To calculate the root-mean square fluctuations (RMSF) for a residue, we first iteratively superimposed the peptides in all MD snapshots to their mean positions by Kabsch's approach (Kabsch, 1976) until the process converges. RMSF of a residue is calculated , where N is the number of heavy atoms in this residue; M is the total number of frames; X i,k is the i-th heavy atom in the k-th frame andX i is the mean position for atom i over all the frames.

Catalytic Activity of the Recombinant Purified Cd-SrtB N26
The recombinant 6xHis-tagged C. difficile sortase enzyme with a deletion of 26 residues at the N-terminal transmembrane domain, designated as Cd-SrtB N26 , was overexpressed in Escherichia coli BL21 (DE3) and purified using an Ni-NTA affinity column (Supplementary Figure 1A). Size exclusion chromatography revealed that Cd-SrtB N26 was eluted at a volume corresponding to an apparent molecular weight of approximately 24 kDa (Supplementary Figure  1B), suggesting that Cd-SrtB N26 exists as a monomer in solution.
To confirm whether the recombinant purified Cd-SrtB N26 retains protease activity, we constructed a fluorescently labeled peptide to observe the fluorescence signal after Cd-SrtB N26 cleaves the substrate peptide in vitro ( Figure 1A). In this construct, the known peptide substrate PPKTG (van Leeuwen et al., 2014) is sandwiched between a fluorophore and quencher. When the peptide remains intact, the intrinsic fluorescence is considerably reduced because of the proximity between the fluorescence donor and quenching acceptor. When the peptide is cleaved, the un-quenched fluorophore gives an enhanced fluorescent signal. To assess whether the previously described sortase inhibitors can inhibit the catalytic activity of Cd-SrtB N26 , MTSET, AAEK1, and curcumin (Maresso et al., 2007;Hu et al., 2013;Donahue et al., 2014) (Supplementary Figure 2) were examined using the fluorescence resonance energy transfer (FRET)-based assay. The concentration-dependent inhibitory effects of MTSET ( Figure 1B), AAEK1 (Figure 1C), and curcumin ( Figure 1D) on the cleavage activity of the recombinant Cd-SrtB N26 were observed as the fluorescence signals were reduced when the inhibitors were added to the reactions comprised of Cd-SrtB N26 and fluorogenic peptides. The results further verified the recombinant Cd-SrtB N26 is catalytically active.

Crystal Structure of Cd-SrtB N26
Cd-SrtB N26 comprises 198 residues with a 6xHis tag at the Cterminus. Cd-SrtB N26 crystallized in space group I23, with the unit cell parameters a = b = c = 121.25 Å and α = β = γ = 90 • . The crystal structure of Cd-SrtB N26 was solved at 2.8 Å resolution by using the molecular replacement method, revealing one molecule in the crystallographic asymmetric unit. Most of the electron density was visible and interpretable for reliable model building. However, the density map for residues 27 and 28, 162-167, 210-216, and 225 as well as the C-terminal 6xHis tag was disordered. The crystallographic data and refinement statistics are summarized in Table 1. Validation of the Cd-SrtB N26 structure by using the MolProbity program  revealed no phi-psi angles in the disallowed region of the Ramachandran map.
Cd-SrtB N26 is structurally equivalent to Cd-SrtB N32,C226A . However, visualizing the sulfhydryl group of the catalytic cysteine residue in Cd-SrtB N26 is essential for facilitating our understanding of the substratespecific catalysis of Cd-SrtB.

In silico Model of the Cd-SrtB N26 -PPKTG Complex
To gain structural insights into how Cd-SrtB N26 recognizes PPKTG, we performed computational modeling based on the crystal structure of the Sa-SrtB-NPQT * complex (PDB 4LFD) (Jacobitz et al., 2014) for predicting the Cd-SrtB N26 -PPKTG structure. In the Sa-SrtB-NPQT * structure, the substratebinding pocket is delineated by a groove near the active site residues within the strands β4 and β7 and within loops β2/β3, β6/β7, and β7/β8. The NPQT * peptide was bound to Sa-SrtB in an "L-shaped" structure via hydrophobic interactions with Leu96, Tyr128, Tyr181, and Ile182 and via hydrogen bonds with Asn92, Thr177, Glu224, and Arg233. Moreover, the almost superimposable hydrophobic residues from S. aureus with the corresponding residues from C. difficile underlies the importance of their function (Supplementary Figure 4) and imply that the Cd-SrtB substrate may be positioned in a similar pattern within the hydrophobic groove. Therefore, we superimposed the structure of Sa-SrtB-NPQT * onto Cd-SrtB N26, mutated NPQT * to PPKT and added a glycine in the C-terminus in silico using the software VMD 1.9.2 (Humphrey et al., 1996) as an initial model of the Cd-SrtB N26 -PPKTG complex. In addition, the missing residues and loops in the Cd-SrtB N26 structure, including the Nand C-terminal residues (27, 28, and 225), and residues located on the β6/β7 (162-167) and β7/β8 (210-216) loops were modeled using UCSF CHIMERA (Yang et al., 2012). To refine the docking pose of the PPKTG in the catalytic pocket of Cd-SrtB N26 , energy minimization and MD simulations were conducted by gradually releasing the restraints on PPKTG, first on the side chains and then on the entire peptide, whereas the Cd-SrtB N26 residues and crystal waters were restrained to their atomic positions in the Cd-SrtB N26 structure at all times during the simulations.
The results from the computational modeling and unrestrained MD simulations suggest that PPKTG stays in the active site, forming a L-shape with a bend toward the N-terminus, resembling the structure of Sa-SrtB-NPQT * complex (Jacobitz et al., 2014) (Figure 3A). The sulfhydryl group of Cys209 is 5.0 Å from the carbonyl carbon of the threonine residue at the P1 position (Schechter and Berger, 1967), which is slightly further apart as compared with that of NPQT * in Sa-SrtB ( Figure 3B). The side chain of Arg217 is hydrogen bonded to the hydroxyl oxygen of P1 Thr. The P2 Lys forms a hydrogen bond with Ser163 and salt-bridge interactions with Asp164. Moreover, the prolyl ring of P4 Pro noncovalently interacts with the aromatic ring of Tyr167. To assess whether Ser163 and Tyr167 are involved in substrate interactions as predicted in the structural model, two mutants that replace Ser163 and Tyr167 with alanine were generated in Cd-SrtB N26 by site-directed mutagenesis. By performing the FRET-based assay, we observed that the cleavage activity of mutants Cd-SrtB N26,S163A and Cd-SrtB N26,Y167A was substantially reduced compared to wild-type Cd-SrtB N26 (Figure 3C). The results indicate that the alanine substitution of Ser163 and Tyr167 did affect the interactions between Cd-SrtB N26 and PPTKG, resulting in the reduced florescence signals. FIGURE 2 | Crystal structure of Cd-SrtB N26 . (A) Ribbon diagram of the SrtB N26 structure comprising three α-helices (H1, H4, and H5), two 3 10 -helices (H2 and H3), and eight β-strands (β1-β8). Helices and β-strands are colored in purple and cyan, respectively. The flexible loops are presented in dashes. (B) Surface representation of Cd-SrtB N26 structure. The catalytic residues Cys209, His116, and Arg217 are colored in yellow, red, and blue, respectively. (C) Part of the overall omit map contoured at 1.0 sigma revealing the side-chain density of the catalytic Cys-His-Arg triad.

Specificity Determinants of Substrate Peptides
To have a better understanding of specific recognition of the substrate peptide PPKTG by Cd-SrtB, we also constructed models of Cd-SrtB N26 -SPKTG, Cd-SrtB N26 -NPKTG, and Cd-SrtB N26 -NVQTG complexes in the same way as constructing model of Cd-SrtB N26 -PPKTG complex. Subsequently, we performed molecular dynamics (MD) simulations to analyze the residues in Cd-SrtB N26 and the contact frequency of those residues with different substrate peptides (Supplementary Figure  5). It is observed that the P4 residues in PPKTG and SPKTG are stabilized by residues of Cd-SrtB N26 located in binding pocket (Tyr101-Arg102, Ser163-Tyr167 for PPKTG, and Asp164-167, Phe213-Asp214 for SPKTG) (Figure 4). Our results show that the P4 residue of PPKTG in Cd-SrtB N26 -PPKTG complex interacts with Asp166 for about 80% of the simulations time (10 ns) and forms hydrogen bonds with Asp166; while the P4 residues in NPKTG and NVQTG do not specifically interact with any residue in Cd-SrtB N26 (Figure 4 and Supplementary Figure 5). Moreover, we assumed that a peptide would be subjected to a conformation that allows Cd-SrtB to achieve a better catalytic efficiency if the distance (DIS Cys-Thr ) between the sulfhydryl group of cysteine residue in Cd-SrtB and the carboxyl carbon of threonine residue in peptide is relatively FIGURE 3 | A computational model of the Cd-SrtB N26 -PPKTG complex. (A) Superimposed structures of the Sa-SrtB-NPQT* and Cd-SrtB N26 -PPKTG models. Blue, Sa-SrtB. Cyan, Cd-SrtB N26 . NPQT* and PPKTG are presented in green and magenta, respectively. (B) The atomic interactions between PPKTG and Cd-SrtB N26 in the structural model. The hydrogen bond is colored in yellow. The distance from the sulfhydryl group of Cys209 to the carbonyl carbon of P1 Thr is represented in red. (C) Catalytic activity of Cd-SrtB N26 with mutation monitored using the FRET-based assay. PPKTG was incubated with recombinant purified SrtB N26 , SrtB N26,Y167A , and SrtB N26,S163A . The increase in the relative fluorescence signal was observed when PPKTG was cleaved by Cd-SrtB N26 . The enzymatic cleavage of Cd-SrtB N26 was inhibited with Y167A and S163A mutations.

DISCUSSION
In this work, we have presented the crystal structure of the catalytically active SrtB from C. difficile and provided a plausible interaction scheme to understand how SrtB recognizes the unique (S/P)PXTG motif. FIGURE 4 | Contact frequency (to the P4 residue of substrate peptides) for residues in Cd-SrtB N26 . Cd-SrtB N26 are shown in surface representation and opaque for the cases of Cd-SrtB N26 -PPKTG (upper-left), Cd-SrtB N26 -SPKTG (upper-right), Cd-SrtB N26 -NPKTG (lower-left) and Cd-SrtB N26 -NVQTG (lower-right). Different peptides are shown as black line, with the P4 residue labeled by ball-and-stick in CPK. The increased contact frequency for the P4 residues in different peptides is colored from blue to red for the purpose of easier visualization.
The P4 residue of the sortase substrate is likely to be the specificity determinant. Based on our computational model of Cd-SrtB N26 -PPKTG complex, we are able to identify that the hydrophobic residue Tyr167 in Cd-SrtB N26 forms specific interaction with P4 Pro in PPKTG, confirmed by sitedirected mutagenesis and FRET-based assay (Figure 3). This hydrophobic interaction between sortase and substrate has not been seen in the current available crystal structures and may be unique to the Cd-SrtB N26 -PPKTG complex. In the structure of Sa-SrtB-NPQT * complex, P4 Asn in NPQT * is hydrogen bonded to the carbonyl backbone of Thr177 within the β6/β7 loop in Sa-SrtB (Jacobitz et al., 2014). The residue Thr177 in Sa-SrtB is structurally equivalent to that of Ser163 in Cd-SrtB N26 . However, structural superposition shows Ser163 in Cd-SrtB N26 is too far away to interact with P4 Asn in Sa-SrtB-NPQT * . Moreover, we also identified that Ser163 interacts with P2 Lys in PPKTG. As Tyr167A and Ser163A mutants exhibited reduced hydrolytic activity (Figure 3), we concluded that these two residues play important roles in specific substrate-binding and that the abolishment of the specific interactions affects the cleavage activity by Cd-SrtB N26 . Taken together, the structural analyses have provided partial explanation why Cd-SrtB N26 does not recognize the NPQTN sorting signal. However, the actual crystal structure of Cd-SrtB-(S/P)PKT * is required to disclose the atomic interactions of the complex.
The simulation studies on the structural models of Cd-SrtB N26 -PPKTG, Cd-SrtB N26 -SPKTG, Cd-SrtB N26 -NPKTG, and Cd-SrtB N26 -NVQTG complexes suggest that the stability of P4 residue may have an effect on the position P1 residue and DIS Cys-Thr (Figures 4, 5). It seems reasonable to imply that the P4 Pro in PPKTG plays a role in configuring FIGURE 5 | The distribution of distances between Cys209 (on SrtB N26 ) and Thr4 (on the peptides) for four examined peptides. Distances between the catalytic Cys209 in Cd-SrtB N26 (upper-left) and Thr4, the P4 residue in the peptides of Cd-SrtB N26 -PPKTG (blue), Cd-SrtB N26 -SPKTG (green), Cd-SrtB N26 -NPKTG (red) and Cd-SrtB N26 -NVQTG (black) of all the snapshots in simulations, are plotted in histogram. Mean distance and deviation for each peptide are provided in the table (upper-right). X-axis is the distance from the sulfur atom of Cys209 to the carboxyl carbon of the Thr in the peptides.
the substrate peptide to a preferred conformation, permitting Cd-SrtB to perform a more efficient cleavage. Furthermore, PPKTG and SPKTG that have a better Cd-SrtB N26 hydrolytic activity than NPKTG and NVQTG peptides (Donahue et al., 2014;Chambers et al., 2015) are found to have comparatively high contacting frequency with Cd-SrtB N26 via their P4 residue and a shorter DIS Cys-Thr throughout the simulations. It suggests the stabilization of P4 residue by surrounding loops near the active site can refrain the mobility of substrate peptides and therefore result in a shorter DIS Cys-Thr prompted for catalysis. The specificity determinant that associates with P4-led peptide conformation provides a molecular basis for specific recognition of PPKTG by Cd-SrtB.

AUTHOR CONTRIBUTIONS
LY, IH, and SW conceived and designed the experiments. JY, CF, YL, JC, and YC performed he experiments. JY, YL, YH, JN, LY, IH, and SW analyzed the data. JY, CF, YL, LY, IH, and SW prepared the manuscript.