The 1.7 Å crystal structure of the C5a peptidase from Streptococcus agalactiae (ScpB) reveals an active site competent for catalysis

A 1.7 Å structure is presented for an active form of the virulence factor ScpB, the C5a peptidase from Streptococcus agalactiae. The previously reported structure of the ScpB active site mutant exhibited a large separation (~20 Å) between the catalytic His and Ser residues. Significant differences are observed in the catalytic domain between the current and mutant ScpB structures resulting with a high RMSDCα (4.6 Å). The fold of the active form of ScpB is nearly identical to ScpA (RMSDCα 0.2 Å), the C5a‐peptidase from Streptococcus pyogenes. Both ScpA and ScpB have comparable activity against human C5a, indicating neither enzyme require host proteins for C5a‐ase activity. These studies are a first step in resolving reported differences in the specificities of these enzymes.


| INTRODUCTION
2][3] These cell envelope proteases (CEPs) contribute to evasion of the host immune system by cleaving the complement factor C5a between residues H67 and K68 abolishing C5a's signaling capability. 2,4ScpA and ScpB are multi-domain subtilases that share 98% amino acid sequence identity, 5,6 yet despite this high sequence identity, ScpA was reported to inactive both human and mouse forms of C5a, 7,8 while ScpB is only effective against human C5a. 3,4This difference in specificity is unexpected as the 19 amino acids that are different between the enzymes are not located in the active site or putative exo-site on the Fn2 domain.Furthermore, the reported structures of an inactive form of ScpB (ScpB mut ) 9 and active ScpA 8 revealed considerable structural differences, including an unusually large separation between the Ser and His catalytic triad residues for ScpB ($20 Å).This led to a proposal that ScpB undergoes conformational Jakki C. Cooney and Todd F. Kagawa contributed equally to this work.changes to adopt an active form.This activation mechanism is not required for ScpA. 8Given the reported discrepancies in the folds, maturation process and specificity of ScpA versus ScpB, we undertook crystallographic studies on the active form of ScpB to clarify the differences reported in the literature.The structure reported herein, suggests that as with ScpA, ScpB is produced as a catalytically competent enzyme and does not require exogenous factors for activity.

| Crystallization, data collection and structure determination
Crystallization conditions for active ScpB were obtained by screening around the conditions reported for ScpA. 8Crystals of ScpB (10.2 mg/mL in 50 mM HEPES/KOH pH 7.5, 100 mM NaCl) were grown from 1:1 mixtures of protein and 2.5 M ammonium sulfate, 0.1 M HEPES/KOH, pH 7.8 at 20 C using the hanging-drop vapordiffusion method.Crystals were flash-cooled in cryoprotectant solution (2.6 M ammonium sulfate, 0.1 M HEPES/KOH, pH 7.6, and 0.8 M sodium malonate).Data collection and final refinement statistics are given in Table 1.
Data were processed and scaled using XDS. 11A molecular replacement solution was obtained with PHASER 12 using the ScpA structure (PDB ID 3EIF) as a search model.The structure was built with COOT, 13 refined with PHENIX 14 and assessed with MOLPROB-ITY. 15The model was evaluated using composite omit maps calculated with CNSSOLVE. 16Paired refinement was used to determine the high-resolution limit of the dataset. 17ordinates were superposed with LSQAB using the CCP4 suite. 18Differences in normalized B-factors (ΔB 0 norm ) were calculated with the BANΔIT toolkit. 19Coordinates and structure factors have been deposited at the PDB (PDB ID 8BTY) and the raw images (https://doi.org/10.51093/xrd-00107)from data collection have been uploaded to the X-Ray Diffraction Archive (XRDa).Figures of structures were generated using PyMOL. 20A B L E 1 Data collection and refinement parameters.
3 | RESULTS AND DISCUSSION

| The ability of ScpB to cleave human C5a is comparable to ScpA
ScpB was produced in an active and stable form that cleaved human C5a (Figure 1A).MS analysis of the product indicate that as with ScpA, ScpB cleaves hC5a between H67 and K68 showing that ScpB C5a-ase activity does not require additional host factors. 92 | The structure of active ScpB is nearly identical to ScpA ScpB crystallized under similar conditions and in the same space group as ScpA (P6 3 22).Following data collection and processing, the structure was refined to final working R and R free of 0.1881 and 0.2161 respectively (Table 1).The asymmetric unit contains 938 protein resi- of the ScpB catalytic triad is well supported in the 2Fo-Fc electron density map (Figure 1C) and form hydrogen bonding interactions expected for active subtilases (Figure 1C).This is consistent with the observed C5a-ase activity for the enzyme (Figure 1A).The structure of active ScpB (Figure 1B) is closer to ScpA (PDB ID 3EIF) than to ScpB mut (PDB ID 1XF1) with RMSDs of 0.2 Å versus 4.6 Å between 934 and 924 Cα atoms (RMSD Cα ) respectively.Figure 1D shows plots of RMSD and normalized atomic displacement factors (ΔB 0 norm ) comparing residues in ScpB with ScpA (black lines) and ScpB mut (colored lines).In both plots, the differences are greater between the ScpB and ScpB mut structures, with the large differences observed for residues in the catalytic domain due to the uncharacteristic fold of ScpB mut .The differences between the catalytic domains of ScpB and ScpB mut structures are illustrated in Figure 1E.Relevant to activity, the most significant structural differences occur in regions with H193 of the catalytic triad, as well as residues in the prime and non-prime sides of the active site (labeled 'i', 'ii' and 'iii' respectively in Figure 1D,E).In contrast to inactive ScpB mut , the helix associated with H193 is buried in catalytic domain of active ScpB allowing proper orientation of the catalytic triad residues.

| Potential role of non-catalytic domains in C5a-peptidase specificity
While the structures of the active forms of ScpA and ScpB are nearly identical, amino acid substitutions occur in the PA and Fn2 domains.Both domains have been implicated in substrate binding and the activity of ScpA. 21,22Thus, given the apparent difference in selectivity reported in the literature for these related proteases, and the host range for GAS and GBS, 23,24 it would be informative to examine the specificity of these enzymes in greater detail.This will support engineering of the C5a-peptidase and allow appropriate selection of animal models for testing the enzymes' role in streptococcal virulence.writingreview and editing; formal analysis; supervision.

AUTHOR CONTRIBUTIONS
dues and 634 water molecules.In addition, 11 sulfate ions, two molecules of malonate, one HEPES molecule, as well as single calcium and sodium ions are observed in the structure.Importantly, the structure F I G U R E 1 Activity and structure of recombinant ScpB.Panel (A) shows cleavage of rhC5a by ScpA and ScpB analyzed by SDS-PAGE.The MS molecular ions are given below each band.The loss of 829.2 Da in the presence of ScpA and ScpB is consistent with inactivation of C5a resulting from cleavage between H67 and K68 in the tail of C5a.Panel B) shows the structure of active ScpB as a cartoon diagram.From N-to C-termini, residues in the catalytic domain ('Cat') are colored salmon, PA domain in blue, Fn1 domain in green, Fn2 domain in cyan, and Fn3 domain in yellow.The catalytic triad residues are shown as red spheres.Panel (C) shows the 2Fo-Fc electron density map of the ScpB catalytic triad residues D130, H193 and S512 contoured at 2σ. Plots of RMSD and differences in normalized B factors (ΔB 0 norm ) are shown in panel (D).ΔB 0 norm values between active ScpB and the 1XF1 structure of ScpB mut are colored green while RMSD values are colored red.The values obtained in comparisons of active ScpB versus ScpA structures are shown as black lines.Differences between the catalytic domains of ScpB and 1XF1 structures are shown in panel (E) with 'putty' representations colored by RMSD.Dashed red lines highlight distances between Cβ atoms (green spheres) of the catalytic triad.Regions with H193 of the catalytic triad, and residues of the non-prime and prime sides of the active site are labeled 'i', 'ii' and 'iii' respectively in panels (D) and (E).