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.
Highlights
Bacterial surface proteins are crucial virulence factors that mediate adhesion to the host as the first step establishing an infection
Size exclusion chromatography revealed that Clostridium difficile sortase B (Cd-sortases. Class B sortases (SrtBs)) 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 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
Summary
Bacterial surface proteins are crucial virulence factors that mediate adhesion to the host as the first step establishing an infection. 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 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). 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)
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