Structural Insights into Substrate Recognition by Clostridium difficile Sortase

Abstract

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.

Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

“…Purified recombinant WT Cd-SrtB and S207A mutant with a deletion of 26 residues at the N-terminal transmembrane region are designated as Cd-SrtB ⌬N26,WT and Cd-SrtB ⌬N26,S207A . The catalytic activity of Cd-SrtB ⌬N26,S207A was assessed by FRET-based assay using PPKTG-containing peptide (Dabcyl-PVPPKTGDSTTIIGE-Edans) as a substrate described previously (40). The results showed that Cd-SrtB ⌬N26,S207A exhibited reduced cleavage activity compared with Cd-SrtB ⌬N26,WT (Fig.…”

“…Crystallographic structures of Cd-SrtB determined by our group (40) and Chambers et al (41) reveal that the overall structure of Cd-SrtB conforms the canonical sortase fold. In addition, our previous study also constructed and validated an in silico model of a Cd-SrtB-PPKTG complex and elucidated the molecular interaction governing the PPKTG recognition (40). It was suggested that all sortases form similar sorting signalbinding grooves.…”

“…The ␤6/␤7 loop plays a substantial role in interacting and discriminating sorting motif because studies showed that the replacement of the ␤6/␤7 loop in Sa-SrtA with the corresponding site from Sa-SrtB results in converting the specificity profile of Sa-SrtA to Sa-SrtB (33). Our previous work has defined two residues located within ␤6/␤7 loop of Cd-SrtB, Ser163 and Tyr167, that are to be in the direct contact with the substrate peptide PPKTG (40). The structural variation in ␤6/␤7 loop is significant between class A and class B sortase.…”

Covalent attachment and Pro‐Pro endopeptidase (PPEP‐1)‐mediated release of Clostridium difficile cell surface proteins involved in adhesion

“…The presence of a single sortase in C. difficile in combination with the set of substrates described above, suggests that this sortase is involved in functions attributed to the housekeeping sortase A in other species. However, structural analysis of the C. difficile sortase demonstrated that it has the highest resemblance with class B sortases (Chambers et al ., ; Yin et al ., ). Additional modeling has been used to elucidate the unique specificity for the Ser and Pro at the first position of the consensus motif, but co‐crystal structures are warranted to explain this in more detail.…”

Disparate subcellular location of putative sortase substrates in Clostridium difficile

“…Moreover, structural insights into how C . difficile sortase specifically recognizes the peptide substrate PPKTG have recently been provided 20 . The protease PPEP-1 (CD2830/Zmp1) has been shown to further cleave substrates CD2831 and CD3246 at a position N-terminal to the CWSS, releasing these proteins into the culture supernatant 19 , 21 , 22 .…”