Crystal structure of pilus-specific sortase from Enterococcus faecalis reveals both open and closed conformations: insights into loop dynamics and substrate recognition

Pilus-specific or class C sortases (SrtCs) catalyze pilus polymerization in Gram-positive bacteria and possess a critical lid near the active site that is in a closed or open conformation. The crystal structure of SrtC, which polymerizes endocarditis and biofilm-associated pili in Enterococcus faecalis, presented here at 1.9 Å resolution (PDB entry 24rr), reveals the flexible lid in both conformations. Two molecules in the asymmetric unit exhibit different lid conformations stabilized by distinct sets of contacts. The DPX motif from the lid at the active site stabilizes the closed conformation, while the TPY motif within the conserved TP(Y/L)XIN(S/T)H motif of the β7-β8 loop from the asymmetric mate pointing towards the active site stabilizes the open conformation. For the first time, proline cis-trans isomerization, likely acting as a molecular switch in SrtC, was captured, regulating the access of secondary substrate to the active-site cleft by toggling between two states at the β7-β8 loop. Lid movement regulates primary-substrate (LPXTG sorting motif) binding and harmonizes conformational changes at the β7-β8 loop for secondary-substrate (YPKN pilin motif) binding. In silico analysis and RP-HPLC-based assays helped to map residues within sorting motifs near the scissile bond, providing insights into enzyme-substrate recognition. Removing the N-terminal lid region enhances E. faecalis SrtC activity. The unprecedented structural snapshots of both open and closed lid conformations, synchronized with the proline switch in the β7-β8 loop, captured in the same crystal structure, advance the understanding of sortase-mediated virulence in enterococci and provide a framework for the development of antivirulence strategies.