Group A Streptococcus (GAS; Streptococcus pyogenes) causes a wide range of infections, including pharyngitis, impetigo, and necrotizing fasciitis, and results in over half a million deaths annually. GAS ScpC (SpyCEP), a 180-kDa surface-exposed, subtilisin-like serine protease, acts as an essential virulence factor that helps S. pyogenes evade the innate immune response by cleaving and inactivating C-X-C chemokines. ScpC is thus a key candidate for the development of a vaccine against GAS and other pathogenic streptococcal species. Here, we report the crystal structures of full-length ScpC wild-type, the inactive mutant, and the ScpC–AEBSF inhibitor complex. We show ScpC to be a multi-domain, modular protein consisting of nine structural domains, of which the first five constitute the PR + A region required for catalytic activity. The four unique C-terminal domains of this protein are similar to collagen-binding and pilin proteins, suggesting an additional role for ScpC as an adhesin that might mediate the attachment of S. pyogenes to various host tissues. The Cat domain of ScpC is similar to subtilisin-like proteases with significant difference to dictate its specificity toward C-X-C chemokines. We further show that ScpC does not undergo structural rearrangement upon maturation. In the ScpC–inhibitor complex, the bound inhibitor breaks the hydrogen bond between active-site residues, which is essential for catalysis. Guided by our structure, we designed various epitopes and raised antibodies capable of neutralizing ScpC activity. Collectively, our results demonstrate the structure, maturation process, inhibition, and substrate recognition of GAS ScpC, and reveal the presence of functional domains at the C-terminal region.

Introduction

Group A Streptococcus (GAS; Streptococcus pyogenes) is a strictly human pathogen that causes a range of infections, from mild conditions, such as pharyngitis and impetigo, to serious infections, such as necrotizing fasciitis, streptococcal toxic shock syndrome, salpingitis, and genitourinary tract infections [1]. Over 750 million GAS infections are reported globally, with 18 million cases of severe GAS infection and half a million deaths annually [24]. GAS infections are prevalent in indigenous communities in the U.S.A., Australia, and Canada [57], and there was a recent outbreak of GAS-associated diseases in England and Wales [8]. Repeated GAS infections may trigger autoimmune diseases, including acute glomerulonephritis, acute rheumatic fever, and rheumatic heart disease. At least 15 million people have chronic rheumatic heart disease and approximately half a million new cases of acute rheumatic fever occur annually [9].

ScpC is one of the most highly up-regulated and conserved virulence factors expressed by GAS [10]. ScpC is a 180-kDa, surface-exposed, subtilisin-like serine protease that acts as an essential GAS virulence factor for the rapid dissemination of bacteria in soft tissues. It acts by cleaving and inactivating interleukin-8 (IL-8) and other C-X-C chemokines [1113], which consequently impair white blood cell migration, and the phagocytosis and degradation of GAS, thus facilitating its spread [10,12,14,15]. Homologs of ScpC are present in more than 200 species/strains of the Streptococcus family, including S. pneumoniae, S. suis, S. thermophilus, S. dysgalactiae, and S. canis [1621].

ScpC is predicted to be secreted and also covalently linked to the streptococcal cell wall via a C-terminal LPXTG cell wall-anchoring motif [13]. ScpC shares sequence similarity with other large multi-domain serine peptidases, and has thus been classified as a cell-envelope proteinase (otherwise known as SpyCEP). CEPs typically have over 1000 residues and possess a signal peptide at the N-terminus followed by a subtilisin-like catalytic domain (Cat domain) with an inserted protease-associated (PA) subdomain, and three to five additional C-terminal domains [22]. ScpC has a multi-domain organization, consisting of a pre-pro (PP) domain for Sec-dependent secretion and autocatalytic activation; a PR domain for catalytic activity; and A and B/H (B-domain and Helical-spacer domain combined) domains of unknown function [23]. The mature ScpC consists of two subunits of 30 and 150 kDa, and both subunits contribute residues to the catalytic triad, composed of Asp151, His279, and Ser617 [24,25]. The N-terminal PR domain mediates the uptake of ScpC into endothelial cells, whereas the PR + A domain is required for IL-8-degrading activity [23]. GAS tissue adherence is supported by matrix proteins, particularly fibronectin and collagen, which are especially attractive targets for bacteria because of their presence in every type of human tissue [26]. Bacterial adhesins that recognize and bind to the extracellular matrix (ECM) belong to the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) protein family [27].

ScpC is ubiquitous in GAS isolates and thus may serve as a potential vaccine candidate [16]. Although the contribution of ScpC to GAS virulence has been extensively studied, little is known about the biochemical properties of the enzyme and its three-dimensional structure. Here, we report the crystal structures of full-length ScpC, its catalytically inactive mutant, and ScpC in complex with an irreversible serine protease inhibitor, AEBSF [4-(2-aminoethyl)-benzenesulfonyl-fluoride-hydrochloride]. Based on these structures, we also modeled the ScpC–IL8 complex and developed antibodies capable of neutralizing ScpC cleavage activity. This study provides details into the structure, maturation process, inhibition, and substrate recognition of ScpC, and highlights the presence of additional C-terminal domains for the possible MSCRAMM function of ScpC.

Materials and methods

Cloning and site-directed mutagenesis

ScpC (Ala34–Ala1613) was cloned into pET32a with an N-terminal histidine tag. The catalytic triad residues, Asp151, His279, and Ser617, were mutated to create single, double, and triple mutants of ScpC. Site-directed mutagenesis was performed using pairs of complementary primers designed with mismatched bases in the middle. PCR was performed with the KAPA HiFi DNA Polymerase, which synthesized the whole plasmid, including the mismatched bases. The methylated wild-type plasmids were digested with 1 µl (1 unit) of Dpn1 FastDigest restriction enzyme for 1 h at 37°C. Mutated plasmids were transformed into Escherichia coli DH5α competent cells and the sequence was verified. The strains and plasmids used in the present study are listed in Supplementary Table S3.

Expression and purification of ScpC and IL-8

The ScpC plasmid was transformed into E. coli BL21 cells and grown in defined M9 medium [28] supplemented with 25 mg/l l-SeMet at 37°C. Protein expression was induced with 250 µM IPTG at 16°C overnight. The protein was purified using a three-step purification process, which includes affinity purification with Ni-NTA beads followed by ion-exchange chromatography and size-exclusion chromatography using Superdex200 column. The fractions were run using SDS–PAGE, and peak fractions were pooled and concentrated to 10 mg/ml. The IL-8 gene was cloned into the pET32a vector with an N-terminal histidine-thioredoxin tag between BamH1 and Xho1 sites. The protein was purified using Ni-NTA chromatography, and the tag was cleaved using PreScission protease overnight at 4°C. After cleavage, the protein was further purified using size-exclusion chromatography on a Superdex75 column.

Protein crystallization

Crystallization screening was conducted by the hanging-drop vapor diffusion method using commercially available crystallization screens from Hampton, Qiagen, and Emerald Biosystems. The crystallization screens were set up at room temperature (25°C) at a concentration of 10 mg/ml. Diffraction-quality crystals for wild-type and mutant ScpC were obtained from an optimized condition of 0.2 M MgCl hexahydrate, 0.1 M Tris, pH 8.65, and 28% PEG3500. The crystals appeared in 4 days. We also carried out co-crystallization experiments for wild-type ScpC with the pefabloc (AEBSF) protease inhibitor from Sigma–Aldrich.

Structure determination

Before data collection, crystals were cryo-protected in reservoir solution supplemented with 25% glycerol and flash-cooled at 100 K. A single-wavelength anomalous dispersion (SAD) dataset for the SeMet-labeled mutant ScpC was collected at the Beamline 13B1 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan, using a Quantum 4R CCD detector [29]. Datasets for the ScpC wild-type (Ala34–Ala1613) and ScpC–inhibitor complex were collected at the Advanced Photon Source of the Argonne National Laboratory (Chicago, IL, U.S.A.) using a Pilatus detector. SeMet-labeled ScpC34–1613 wild-type crystals diffracted to 2.8 Å. Data processing was done using the HKL2000 program [30]. For the ScpC double mutant, there were two molecules in the asymmetric unit and the crystals belonged to the C21 space group. The Matthews coefficient was estimated to be 2.62 Å3/Da [31], corresponding to a solvent content of 53%. For the native ScpC and ScpC–AEBSF complex, there was only one molecule in the asymmetric unit and the crystals belonged to the P6222 space group. The Matthews coefficient was estimated to be 3.93 Å3/Da, corresponding to a solvent content of 68%. The ScpC crystals were run using SDS–PAGE gel and the size of the full-length protein as the starting material was confirmed.

The initial SAD phases were obtained for the ScpC double mutant using the Crank program [31], and the model was built using the AutoBuild program [32], followed by several rounds of manual model building using COOT [33]. The structure was refined using Phenix-refine [34]. The model was well refined and had good stereochemistry, with more than 99% of the residues within the allowed regions of the Ramachandran plot, as analyzed by PROCHECK (Supplementary Figure S1) [35]. The wild-type and ScpC–AEBSF complex structures were determined by the molecular replacement method using the Phenix-Phaser program [36]. The mutant ScpC structure was used as the model. Despite the disordered N- and C-terminal residues, the electron density map for the residues from Lys115 to Lys1574 of ScpC was well defined in almost all regions, including across the three catalytic residues (Supplementary Figure S2). However, the electron density was not well defined for residues Phe214–Asp272, which harbors the autocatalytic cleavage site (Gln244/Ser245). There were three calcium ions and two sulfate ions bound to the ScpC molecule in the wild type.

Generation of polyclonal antibodies from rabbit

After analyzing the crystal structure of ScpC, epitopes were identified using various fragments of the active site. Three epitopes were identified: PR domain, short fragment of ScpC (Val122–Asn381), and peptide B (21 aa; DDDTKYESHGMHVTGIVAGNC). The ScpC PR domain and the short fragment of ScpC (Val122–Asn381) were cloned, expressed, and purified in our laboratory, whereas peptide B was synthesized by Genscript. Rabbit polyclonal antibodies were generated by Genscript by injecting 0.2 mg of the structurally identified epitopes (with complete Freund's adjuvant) into 3.5-month-old male, New Zealand White Rabbits for immunization. Subsequent booster doses were given at 15-day intervals (with incomplete Freund's adjuvant). After the second and third immunizations, a test bleed was taken to check the antibody titers. At the end of the third immunization, animals were sacrificed and the antibodies were affinity purified. All three polyclonal antibodies could recognize the full-length ScpC (Supplementary Figure S6).

IL-8 cleavage activity assay

Wild-type, double-mutant, and PR + A constructs of ScpC were mixed with IL-8 in an equimolar ratio (20 µM each) in a final reaction volume of 50 µl in a buffer containing 20 mM Tris–HCl, pH 7.5, 200 mM NaCl, and 5% glycerol. The samples were incubated for 2 h at 37°C and then run on 15% SDS–PAGE gel to check for IL-8 cleavage. To test the blocking efficiency of the antibodies, ScpC was pre-incubated with each of the three antibodies and then subjected to the same IL-8 cleavage assay described above. ScpC alone, ScpC treated with the pefabloc inhibitor, and native IL8 were used as controls.

Modeling of ScpC–IL8 complex

We modeled the ScpC–IL8 complex using residues Ser116–Lys1147 of ScpC (wild-type) and the previously published human IL8 model (PDB code: 5D14). Modeling of the ScpC–IL8 complex was carried out using the ROSETTADOCK program [37]. The catalytic triad residues of ScpC were used to guide the identification of the potential binding region of IL-8.

Results

Structure of ScpC

The structures of ScpC34–1613 wild type and the catalytically inactive double mutant (His279A, Ser617A) were determined at 2.8 and 3.1 Å resolution, respectively (Figure 1 and Table 1). In the crystallized constructs (residues: Ala34–Ala1613, 174 kDa), the signal peptide sequence (residue: M1-A33) and the C-terminal anchoring domain (residues: Leu1614–Asp1647) were absent. The structure of ScpC showed nine independent domains (Figure 1A,B) across residues 116–1574. The PP domain (residues: Ala34–Lys115) and part of the W domain (residues: Val1575–Ala1613) are disordered and hence not modeled. To our knowledge, this is the largest structure reported for any member of the subtilisin family. Domain-1 and part of domain-3 fold to form the core catalytic domain (Cat domain), with domain-2 (PA domain) positioned away from this catalytic region. Four β sheets and two α helices from domain 1 and domain 3 form the active-site cleft. According to previous studies [23], only the PR + A region is necessary for IL-8 cleavage activity. Here, we have clarified that this functionally active region of ScpC consists of the Cat, PA, Fn1, Fn2, and Fn3 domains (Figure 1C and Supplementary Figure S3). The first five domains (Ser118–Thr1144) are structurally similar to those found in subtilisin-like proteases but with significant differences that determine the specificity of ScpC toward the C-X-C chemokines. The last four domains (B/H domain and W domain: resi Lys1145–Lys1576) are unique to ScpC (Figure 1C, and Supplementary Figure S3 and Table S1) and comprise a fibronectin/Indian hedgehog protein (Fn4) domain, a reverse-Ig fold, a collagen-binding/pilin domain, and a cell-adhesion domain (Figure 1C,D).

Structure of ScpC.

Figure 1.
Structure of ScpC.

(A) Overall structure of ScpC showing the nine domains. The N- and C-termini are marked, and the domains of ScpC are color-coded and numbered. (B) Topology diagram of ScpC. (C) Domain organization of ScpC. All nine domains are numbered, and the sequence boundaries are indicated. (D) The structure of the individual domains of ScpC. All nine domains follow the same color coding as in (A).

Figure 1.
Structure of ScpC.

(A) Overall structure of ScpC showing the nine domains. The N- and C-termini are marked, and the domains of ScpC are color-coded and numbered. (B) Topology diagram of ScpC. (C) Domain organization of ScpC. All nine domains are numbered, and the sequence boundaries are indicated. (D) The structure of the individual domains of ScpC. All nine domains follow the same color coding as in (A).

Structure of the ScpC–inhibitor complex

Sulfonyl-fluorides are well-known inhibitors of serine proteases [38], and previous studies have reported that ScpC activity can be inhibited by the serine protease inhibitor Pefabloc (AEBSF) [13], but is unaffected by various other protease inhibitors, including other serine protease inhibitors, such as PMSF, aprotinin, and bestatin. Based on these observations, we sought to understand this specific inhibition of ScpC by AEBSF. The structure of ScpC34–1613 in complex with the AEBSF inhibitor was solved at 3.0 Å resolution (Table 1). We show that the fluorosulfonyl terminus of the AEBSF inhibitor is linked covalently via a sulfonyl ester bond to the active-site Ser617 Oγ (Figure 2A,B), which inhibits ScpC activity. Binding of this inhibitor also causes movement of the active-site His279 residue, which breaks the hydrogen bond between His279 and Asp151, that is essential for catalysis (Figure 2A). The NE2 atom of His279 makes a hydrogen bonding contact with the O2S of AEBSF, and the ND2 atom of the oxyanion-hole residue Asn381 also interacts with AEBSF. A hydrophobic cluster is formed by the side chains of Met618, Ser346, Phe579, Thr614, Gly615, and Thr616 of ScpC along with the inhibitor AEBSF. Moreover, the tail of AEBSF (N8) interacts with Arg965 from the Fn2 domain of ScpC.

Structure of ScpC–AEBSF complex.

Figure 2.
Structure of ScpC–AEBSF complex.

(A) Surface representation of the catalytic site of ScpC (gray) with a bound AEBSF molecule (stick representation). The substrate-binding sites and catalytic site residues are shown in cyan and labeled. (B) The feature-enhanced map (2FoFc map) showing AEBSF bound to Ser617 of ScpC contoured at 1σ. The fluorosulfonyl terminus of the AEBSF inhibitor is linked covalently via a sulfonyl ester bond to the active-site Ser617 Oγ.

Figure 2.
Structure of ScpC–AEBSF complex.

(A) Surface representation of the catalytic site of ScpC (gray) with a bound AEBSF molecule (stick representation). The substrate-binding sites and catalytic site residues are shown in cyan and labeled. (B) The feature-enhanced map (2FoFc map) showing AEBSF bound to Ser617 of ScpC contoured at 1σ. The fluorosulfonyl terminus of the AEBSF inhibitor is linked covalently via a sulfonyl ester bond to the active-site Ser617 Oγ.

Table 1
Data collection and refinement statistics.
 ScpC_wild type ScpC_mutant ScpC_complex 
Data collection 
 Space group P6222 C121 P6222 
 Cell dimensions 
  a, b, c (Å) 191.63, 191.63, 250.96 182.13, 132.85, 151.99 190.53, 190.53, 248.67 
  α, β, γ (°) 90, 90, 120 90, 100.69, 90 90, 90, 120 
 Resolution (Å) 50.0–2.8 (2.9–2.8) 50–2.93 (2.98–2.93) 50–2.8 (2.87–2.8) 
Rmerge 0.21 (0.61) 0.19 (0.72) 0.17 (0.91) 
I/σI 17.5 (2.0) 7.8 (1.4) 13.3 (1.4) 
 Completeness (%)1 99.9 (99.8) 98.4 (82.8) 99.4 (91.5) 
 Redundancy1 18.6 (8.4) 5.2 (2.3) 27.7 (4.2) 
Refinement 
 Resolution (Å) 19.9–2.8 (2.9–2.8) 19.9–3.1 (3.21–3.1) 19.9–3.0 (3.03–3.0) 
 No. of reflections 66 560 (6453) 64 125 (6364) 55 238 (3345) 
Rwork/Rfree 0.207/0.257 0.205/0.264 0.223/0.278 
 No. of atoms 
  Proteins 10 537 19 810 10 279 
  Ligand/ion 15 15 29 
  Water 40 
B-factors (Å2
  Proteins 68.30 61.38 42.35 
  Ligand/ion 69.65 83.77 58.80 
  Water 48.9   
 R.m.s deviations 
  Bond lengths (Å) 0.012 0.012 0.013 
  Bond angles (°) 1.34 1.35 1.42 
  PDB code 5XYR 5XXZ 5XYA 
 ScpC_wild type ScpC_mutant ScpC_complex 
Data collection 
 Space group P6222 C121 P6222 
 Cell dimensions 
  a, b, c (Å) 191.63, 191.63, 250.96 182.13, 132.85, 151.99 190.53, 190.53, 248.67 
  α, β, γ (°) 90, 90, 120 90, 100.69, 90 90, 90, 120 
 Resolution (Å) 50.0–2.8 (2.9–2.8) 50–2.93 (2.98–2.93) 50–2.8 (2.87–2.8) 
Rmerge 0.21 (0.61) 0.19 (0.72) 0.17 (0.91) 
I/σI 17.5 (2.0) 7.8 (1.4) 13.3 (1.4) 
 Completeness (%)1 99.9 (99.8) 98.4 (82.8) 99.4 (91.5) 
 Redundancy1 18.6 (8.4) 5.2 (2.3) 27.7 (4.2) 
Refinement 
 Resolution (Å) 19.9–2.8 (2.9–2.8) 19.9–3.1 (3.21–3.1) 19.9–3.0 (3.03–3.0) 
 No. of reflections 66 560 (6453) 64 125 (6364) 55 238 (3345) 
Rwork/Rfree 0.207/0.257 0.205/0.264 0.223/0.278 
 No. of atoms 
  Proteins 10 537 19 810 10 279 
  Ligand/ion 15 15 29 
  Water 40 
B-factors (Å2
  Proteins 68.30 61.38 42.35 
  Ligand/ion 69.65 83.77 58.80 
  Water 48.9   
 R.m.s deviations 
  Bond lengths (Å) 0.012 0.012 0.013 
  Bond angles (°) 1.34 1.35 1.42 
  PDB code 5XYR 5XXZ 5XYA 
1

Values in parentheses are for highest-resolution shell.

Comparison of the ScpC wild-type, mutant, and inhibitor complex

A comparison of wild-type, inactive double-mutant, and inhibited forms of ScpC structures revealed that they align very well (r.m.s.d. = 0.9 Å for 1297 Cα atoms) (Supplementary Figure S4). For wild-type ScpC, the active-site serine residue (Ser617) makes a hydrogen bonding contact with the active-site His279, which, in turn, forms a hydrogen bonding contact with Asp151. Both wild-type and double-mutant structures superpose well, and the catalytic triad residues remain in their original positions (Figure 3A). However, the residues between Gly599 and Ser612 form an antiparallel β-sheet in the wild type and in the complex, but are disordered in the mutant protein. We speculate that these well-ordered secondary structures close to the active-site region in the wild-type and protein–inhibitor complex play a role in the substrate binding. Moreover, we observed that the side chains of residues His279 and Ser617 move subtly in the inhibited form of ScpC when compared with the wild-type protein. The side chain of Met618, which is adjacent to the active-site Ser617, also moves 2.5 Å in the inhibited form of ScpC (Figure 3B). Besides, in the C-terminal domain-5 (Fn3), there is significant movement of the Cα atoms in the loop region of wild-type ScpC (Pro1123–Leu1135) of up to 5 Å when compared with the double mutant. Zingaretti et al. [24] reported that the catalytic mutants of ScpC have comparable thermal stability as wild-type proteins.

Structural comparison of the active-site regions of ScpC.

Figure 3.
Structural comparison of the active-site regions of ScpC.

(A) Structural comparison of the active-site region between ScpC wild type (brown) and ScpC double mutant (pink). The catalytic triad residues are in the same positions. (B) Structural comparison of the active-site region between ScpC wild type (brown) and ScpC inhibited (blue). There is subtle movement of the side chains of the active-site residues His279 and Ser617 in the inhibited ScpC. The side chain of M618, which is adjacent to Ser617, moves 2.48 Å in the ScpC inhibited form when compared with the wild type.

Figure 3.
Structural comparison of the active-site regions of ScpC.

(A) Structural comparison of the active-site region between ScpC wild type (brown) and ScpC double mutant (pink). The catalytic triad residues are in the same positions. (B) Structural comparison of the active-site region between ScpC wild type (brown) and ScpC inhibited (blue). There is subtle movement of the side chains of the active-site residues His279 and Ser617 in the inhibited ScpC. The side chain of M618, which is adjacent to Ser617, moves 2.48 Å in the ScpC inhibited form when compared with the wild type.

Comparison of Cat domain of ScpC with homologs

A structural homology search of the full-length ScpC structure consisting of all nine domains revealed no structural homologs. However, the Cat domain of ScpC has a subtilisin-like fold similar to that of C5a peptidases ScpA and ScpB (Supplementary Table S1). Although the catalytic pockets are conserved among ScpC, ScpB, and ScpA, the His and Ser residues of the catalytic triad of ScpC are ∼86 residues away in linear sequence toward the C-terminus compared with the sequences of ScpA and ScpB (Supplementary Figure S5). This is due to the presence of a unique, flexible region in ScpC, spanning residues Ser206–Glu275, which is replaced by a small, 11-residue loop in ScpA and ScpB. Comparing the Cat domains of ScpC, ScpA, and ScpB, we found that ScpC resembles the active ScpA but is significantly different from the inactive ScpB (Supplementary Figure S6).

Notably, only ScpC is capable of cleaving the human C-X-C chemokines, whereas ScpA and ScpB have different substrate specificities [39]. A comparison of the ScpC and ScpA active-form structures identified variations in several regions in the Cat, Fn1, Fn2, and Fn3 domains (Supplementary Figure S7). Given that the Fn2 and Fn3 domains are reported to be potential docking sites for the substrate [39], the structural differences in these regions might be related to the substrate recognition of ScpC.

No structural rearrangement upon maturation of ScpC

ScpC undergoes a unique autocatalytic cleavage between residues Gln244 and Ser245 to become an active protease. This is different from other C5a peptidases, which are not cleaved for maturation. We found that the autocatalytic cleavage of ScpC was unaffected by mutating Gln244Ala (Figure 4A), and that the ScpC Gln244Ala mutant retains both the autocatalytic and IL-8 cleavage capabilities (Figure 4B). On the other hand, mutating a single, catalytic triad residue abrogated the autocatalytic cleavage and catalytic activity of ScpC (Figure 4C). Similar results were observed in previous studies [24].

Maturation of ScpC.

Figure 4.
Maturation of ScpC.

(A) Autocatalytic cleavage is unaffected by Q244A mutation. SDS–PAGE gel showing two bands of ScpC with the Q244A mutation. (B) IL-8 cleavage assay using the ScpC (Q244A) mutant showed that this mutant retains IL-8 cleavage activity (Top). The cleavage assay was quantified by plotting loss of IL-8 for each lane. Assays were carried out in triplicates (Bottom). Western blot shows that the Q244A mutant can cleave IL-8. (C) SDS–PAGE gel showing a single band of ScpC (Ser617A). The single mutation of the catalytic triad residue abolishes the autocatalytic cleavage activity of ScpC. (D) The PR domain can carry out the autocatalytic cleavage. SDS–PAGE gel showing two bands of ScpC due to autocatalytic cleavage.

Figure 4.
Maturation of ScpC.

(A) Autocatalytic cleavage is unaffected by Q244A mutation. SDS–PAGE gel showing two bands of ScpC with the Q244A mutation. (B) IL-8 cleavage assay using the ScpC (Q244A) mutant showed that this mutant retains IL-8 cleavage activity (Top). The cleavage assay was quantified by plotting loss of IL-8 for each lane. Assays were carried out in triplicates (Bottom). Western blot shows that the Q244A mutant can cleave IL-8. (C) SDS–PAGE gel showing a single band of ScpC (Ser617A). The single mutation of the catalytic triad residue abolishes the autocatalytic cleavage activity of ScpC. (D) The PR domain can carry out the autocatalytic cleavage. SDS–PAGE gel showing two bands of ScpC due to autocatalytic cleavage.

Despite the structural similarity of ScpA and ScpB to the ScpC Cat domain, these C5a peptidases do not undergo autocatalytic cleavage for maturation. However, significant conformational differences are observed in the catalytic cleft between the active ScpA and the mutant ScpB (r.m.s.d.: 6.3 Å for 328 Cα-atoms) [39]. Similarly, other well-known proteases, like thrombin and trypsin, undergo multiple rounds of cleavage and structural rearrangement to become active proteases [40,41]. For ScpC wild-type (active form), we show that the N-terminal (Leu119–Gln213) and the C-terminal (Asp273–Asp1577) fragments are held together by several hydrogen bonds (Supplementary Table S2) and hydrophobic interactions. However, the electron density was not well defined for any of the three structures for modeling the region consisting of residues Phe214–Asp272, which harbors the autocatalytic cleavage site (Gln244/Ser245) (Supplementary Figure S3B). Furthermore, in ScpC, we found no major structural differences between the inactive mutant and the active wild type (Figure 3A and Supplementary Figure S4), indicating that no structural re-arrangement occurs upon maturation of ScpC. This observation is similar to that for chymotrypsin, where no major structural changes were observed upon maturation [42]; albeit, chymotrypsin is a smaller protease (245 residues) compared with ScpC.

PR domain is functionally active

It has been previously reported that the PR + A domain is required for IL-8-degrading activity [23]. Here, using the three-dimensional structure of ScpC, we showed that the PR domain has all the catalytic triad residues and can fold properly to make an active protease. Based on this observation, we generated a construct consisting of only the PR domain (Cat + PA + Fn1), and we observed that this construct can undergo autocatalytic cleavage (Figure 4D). Thus, we propose that the A domain of ScpC is only required for substrate recognition and binding, whereas the PR domain alone is a functional protease.

ScpC cleaves its substrate IL-8 protein between residues Gln59 and Arg60 [13]. Our brief attempt did not yield diffraction-quality crystals, and hence, we modeled the complex using ROSETTADOCK [37] to understand the interaction between ScpC and IL-8 (Figure 5A,B). We found that the C-terminal region of IL-8 is buried in the catalytic cleft of ScpC. The scissile bond is positioned close to the Ser617 Oγ, with the His279 side-chain nitrogen atoms (NE2 and ND1) within H-bonding distance to the side chains of Ser617 and Asp151. The oxyanion-hole residue Asn381 is located at H-bonding distance with Ser617 (Figure 5A,B). Residues Phe960–Lys968 form an extended loop in the Fn2 domain and interact with IL-8 to stabilize the substrate in the catalytic cleft. Residues Ser346/Leu347/Gly348 in ScpC — which are thought to recognize the substrate backbone in the nonprime region of the active site [39] — make hydrogen bonding contacts with IL-8.

ScpC–IL-8 complex model.

Figure 5.
ScpC–IL-8 complex model.

(A) Surface representation of ScpC showing a bound IL-8 molecule. Cartoon representation of IL8, bound to the catalytic site, is shown in magenta. Color code: ScpC PR domain — magenta; A domain — green and B/H domain — blue. (B) The zoomed view of the interacting region. The catalytic triad residues are labeled and shown as stick representation. Cleavage occurs between residues Q59 and R60 (stick representation) of IL-8. The model was generated using the ROSETTADOCK program.

Figure 5.
ScpC–IL-8 complex model.

(A) Surface representation of ScpC showing a bound IL-8 molecule. Cartoon representation of IL8, bound to the catalytic site, is shown in magenta. Color code: ScpC PR domain — magenta; A domain — green and B/H domain — blue. (B) The zoomed view of the interacting region. The catalytic triad residues are labeled and shown as stick representation. Cleavage occurs between residues Q59 and R60 (stick representation) of IL-8. The model was generated using the ROSETTADOCK program.

Notably, the Cat, PA, and Fn domains have been identified by others to play key roles in substrate recognition in the homologs [39,43]. In the C-terminal domain-5 (Fn3), which is part of A domain, there is up to 5 Å movement of Cα atoms in the loop region of wild-type ScpC (Pro1123–Leu1135) as compared with the double-mutant ScpC. Furthermore, the disordered regions located in the PR domain (Gly598–Gln613) of the double mutant and wild type that are close to the active-site Ser617 are well defined in the ScpC–AEBSF complex, suggesting that these flexible regions might be important for the catalytic activity. Notably, the Cat, PA, and Fn domains have been identified by others to play key roles in substrate recognition in the homologs [39,43].

ScpC structure reveals the presence of multiple MSCRAMM domains

A structural homology search for the unique domains in the C-terminal region of ScpC (Asp1148–Gly1574, domains 6–9; Supplementary Figure S3 and Table S1) revealed that domain 6 has a fibronectin fold, similar to domains 3, 4, and 5. Domain 7 of ScpC shows similarity to the major histocompatibility complex heavy chain, whereas domain 8 is similar to pilins and collagen-binding proteins, particularly collagen adhesion protein and cell wall surface anchor protein. This domain also shows structural homology with several major pilins from Gram-positive bacteria, including S. pyogenes [44]. Domain 9 of ScpC is structurally similar to cell-adhesion proteins, β-amylases, lipases, and lipoxygenases. Lipase is associated with bacterial virulence and causes significant reductions in bacterial loads when mutated [45].

MSCRAMMs are a group of proteins that shares common structural features. MSCRAMMs have an N-terminal signal sequence, a variable N-terminus, followed by one to many ECM-binding regions, and an LPxTG sequence motif for attaching to the bacterial surface [46]. ScpC protein has all the above features, and hence, it falls under this category of proteins. Especially its C-terminal region has many different types of ECM-binding protein domains, namely fibronectin III domain (four repetitive units), collagen-binding domain, and pilin-like domain (Figure 1C). A close structural homolog of ScpC from Group B Streptococcus, ScpA, is also characterized as an MSCRAMM [47].

Targeting ScpC activity using antibodies

Taking clues from the structure of ScpC, particularly the Cat domain, we raised rabbit polyclonal antibodies against three epitopes of ScpC: the PR domain, a short fragment of ScpC (Val122–Asn381), and peptide B (Asp271–Asn290). The short fragment of ScpC (Val122–Asn381) includes the flanking regions of the catalytic core and two catalytic triad residues, whereas peptide B was selected based on its position in the catalytic cleft. All three antibodies recognized the full-length ScpC (Supplementary Figure S8). We then assessed whether these antibodies could protect human chemokines from ScpC cleavage using IL-8 as a representative protein. ScpC was pre-incubated with each antibody for 1 h at room temperature and then subjected to IL-8 cleavage assay. We found that, for all three antibodies, the antibody-bound ScpC was unable to cleave IL-8 when compared with the unbound wild-type ScpC (Figure 6). Thus, all three antibodies successfully blocked the chemokine cleavage activity of ScpC. It is interesting to note that the antibodies raised using the short peptide were sufficient to block the activity. Further optimization of this peptide fragment could lead to improved, short peptides that could be used as potential vaccine candidates against ScpC.

Cleavage of IL-8 by ScpC in the presence of various ScpC-specific antibodies.

Figure 6.
Cleavage of IL-8 by ScpC in the presence of various ScpC-specific antibodies.

(A) The cleavage efficiency of ScpC was tested in the presence of various antibodies using IL8 as substrate. Anti-IL-8 antibody was used to detect the bands using western blot. The lane details are explained in the figure. (B) The cleavage assay was quantified by plotting loss of IL-8 for each lane in (A). Assays were carried out in triplicates.

Figure 6.
Cleavage of IL-8 by ScpC in the presence of various ScpC-specific antibodies.

(A) The cleavage efficiency of ScpC was tested in the presence of various antibodies using IL8 as substrate. Anti-IL-8 antibody was used to detect the bands using western blot. The lane details are explained in the figure. (B) The cleavage assay was quantified by plotting loss of IL-8 for each lane in (A). Assays were carried out in triplicates.

Discussion

Virulence factors are up-regulated in invasive GAS isolates and this distinguishes them from most colonizing strains [10]. ScpC is a major GAS virulence factor and enables bacterial spread in soft tissues by impairing the functions of neutrophils in the primary host defense response against GAS infections [48]. It also serves as a key candidate for vaccine development against GAS and other pathogenic streptococcal species [16,19,20,4952].

We have determined the structures of ScpC (wild-type), inactive-mutant ScpC, and the ScpC–inhibitor complex. The structure of the full-length ScpC protein consists of Cat domain, PA domain, Fn domains (PR + A domains) and additional collagen-binding, cell-adhesion, and pilin-like domains, which might be responsible for MSCRAMM function. These additional domains have not been predicted in previous reports, and are not present in other C5a peptidases, which bear structural similarity only to the first five domains of ScpC. We have also identified that the N-terminal PR + A domain, which is essential for IL-8 cleavage activity, consists of Cat, PA, Fn1, Fn2, and Fn3 domains.

We further explored the autocatalytic cleavage action of ScpC. Intriguingly, we found that, although cleavage occurs between residues Gln244 and Ser245, the protein retains its autocatalytic function even after mutation of these residues. In the structure, we observed that the region spanning residues Gln213 to Asp273, which includes the autocatalytic site, is completely flexible, and this points to the possibility of multiple sites of autocatalytic cleavage in this region. Yet, our studies and previous reports [24] show that a single mutation of a catalytic triad residue abrogates autocatalytic cleavage and the enzymatic activity of the protein. We show that the PR + A domain is essential for both of these functions. In addition, we show that the PP region, comprising residues 1–123, is also flexible, and that the N-terminal fragment (Leu119–Gln213) and the C-terminal fragment (Asp273–Asp1577) are held together by several hydrogen bonds and hydrophobic interactions. There were no major structural changes observed between the inactive mutant and the active wild type, indicating that no structural re-arrangement occurs upon maturation of ScpC. Overall, these findings point to quite a unique autocatalytic cleavage mechanism of ScpC. A similar autocatalytic cleavage mechanism has been shown previously for chymotrypsin, which undergoes two autolytic cleavages that result in the loss of four amino acids resulting in the formation of three polypeptide chains in the mature enzyme [53]. These three chains are held together by two inter-chain disulfide bonds. However, in ScpC, the active protease comprises a PP region and two fragments held together by several hydrogen bonds.

The structure of ScpC in complex with the protease inhibitor pefabloc (AEBSF) revealed the mechanism of inhibition at the atomic level. Through an analysis of the complex structure, we identified the various points of contact between the inhibitor and the protein residues, specifically the unique interactions between the tail of AEBSF (N8) with Arg965 from the Fn2 domain of ScpC and the formation of the hydrophobic cluster. This knowledge will aid in the design of next-generation inhibitors that could be further developed into drug-like molecules that target the activity or function of ScpC [54]. Indeed, a similar approach was used in the development of protease inhibitors as drug against HIV-1 and hepatitis C viruses [5557].

Glycosaminoglycans (GAGs) are complex, linear polysaccharides of the ECM [58]. IL-8 binds avidly to GAGs and this binding modulates neutrophil recruitment [59,60]. We show that ScpC possesses pilin and lipase-like domains that potentially mediate ScpC binding to the ECM on host cells. These domains probably evolved to ensure cleavage and inactivation of all forms of IL-8, i.e. free in solution and surface bound. Kaur and others previously reported that ScpC can mediate its own uptake as a secreted protein in human endothelial cells [23]. ScpC also plays a key role in bacterial dissemination through soft tissues and is implicated in the spread of the bacteria from the nasopharynx to the lung. Recent studies have shown that ScpC impedes bacterial clearance and aids bacterial dissemination [15]. The adhesion of Streptococci to the ECM is an essential step in the infection cascade, with various ECM proteins, such as collagen, fibrinogen, laminin, vitronectin, or fibronectin present on the host cell surface [61]. Based on our structural analysis, we hypothesize that the additional fibronectin and collagen-binding domains of ScpC will help in its competitive binding to ECM proteins like integrin and collagen. Thus, our ScpC structure unraveled the hitherto unknown function of the A and B/H domains (C-terminal domains 6–9) of ScpC.

Viral infections, such as Influenza, Varicella, Morbilla, and Epstein–Barr Virus, can increase host susceptibility to bacterial infections [62], presumably because viral neuraminidases remove sialic acid from respiratory cells, which can increase bacterial adherence [63,64]. We speculate that ScpC will play an active role in this altered environment as a bacterial adhesion protein which increases the bacterial adherence of S. pyogenes to the host cells.

Overall, the present study sheds light on the mechanism of ScpC activity and this knowledge will be of paramount importance in the development of therapeutic strategies against GAS infections.

Data availability

Co-ordinates of ScpC wild-type, ScpC double-mutant, and ScpC–AEBSF complex structures have been deposited in the Protein Data Bank under accession codes 5XYR, 5XXZ, and 5XYA.

Abbreviations

     
  • AEBSF

    4-(2-aminoethyl)-benzenesulfonyl-fluoride-hydrochloride

  •  
  • CEP

    cell-envelope proteinase

  •  
  • ECM

    extracellular matrix

  •  
  • GAGs

    glycosaminoglycans

  •  
  • GAS

    Group A Streptococcus

  •  
  • IL-8

    interleukin-8

  •  
  • MSCRAMMs

    microbial surface components recognizing adhesive matrix molecules

  •  
  • PA

    protease-associated

  •  
  • PP

    pre-pro

  •  
  • SAD

    single-wavelength anomalous dispersion

Author Contribution

J.S. and E.H. conceived the study. C.J., D.B., E.H., and J.S. designed the experiments. C.J., T.Y.C., M.T.P., and D.N. performed experiments. N.S.P. and C.J. phased the structure. C.J. and J.S. analyzed the data. C.J., E.H., and J.S. wrote the manuscript with input from D.B.

Funding

This work was partially supported by the Ministry of Education Singapore MoE-T2 grant [WBS R154-000-625-112] and NUS AcRF T-1 grant [R154-000-683-112] Singapore given to J.S. E.H. acknowledges the support by the National Research Foundation, Prime Minister's Office, Singapore under its Campus of Research Excellence and Technological Enterprise (CREATE) programme. Crank software development is supported by grant 13337 to N.S.P. from the Stichting voor de Technische Wetenschappen (STW).

Acknowledgments

The data reported in the present study are collected at the National Synchrotron Radiation Research Centre (NSRRC, Taiwan) Beamline 13B1 and NECAT Beamline 24IDC at Advanced Photon source, Argonne National Laboratory, U.S.A. NECAT beamlines, are funded by the NIGMS from the National Institutes of Health [P41 GM103403]. The Pilatus 6M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant [S10 RR029205]. Advanced Photon Source, is a U.S. (DOE) User Facility operated by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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