Mechanistic Insights into ComC Peptide Recognition by the Peptidase Domain of ComA in Streptococcus mutans for Quorum...

In Streptococcus mutans, the recognition and cleavage of a 46-residue precursor peptide known as ComC by the peptidase (PEP) domain of the ComA protein trigger quorum-sensing-mediated biofilm formation, which is a key factor in dental caries. The mechanism behind the high fidelity of ComC processing by the PEP domain remains unclear primarily due to the lack of structural characterization of the ComC:PEP complex. We present a model of the precatalytic complex where a truncated 18-residue ComC peptide is bound to the PEP. In this model, a protonated histidine residue at the active site stabilizes the complex. Additionally, the conserved residues of the peptide, F11 and L19, are situated in the hydrophobic cleft of the protein, providing a plausible explanation for their evolutionary conservation. Using the 18-ComC:PEP complex as a template, we quantitatively assessed the changes in PEP binding affinity (ΔΔG) resulting from alanine mutations at various hydrophobic residues within the 18-ComC peptide. Our findings demonstrate that the energetics of peptide-protein interactions significantly varies based on the location of the alanine mutations. The PEP protein showed strong selectivity against alanine substitutions at conserved positions in 18-ComC, with a ΔΔG of approximately 2.5 kcal/mol, supporting previous kinetic experiments. The simulations also identified three weakly selective positions (ΔΔG < 0.5 kcal/mol) within the 18-ComC that could be targeted for future substrate-based peptide inhibitor design. The alanine substitution in the Gly-Gly motif present before the cleavage site in 18-ComC disrupted the precatalytic state by preventing the accommodation of a methyl group within the PEP protein. The simulations demonstrate the importance of the Gly-Gly signal sequence for ComA recognition and cleavage in experimental assays. Simulations with the postcatalytic complex indicated that deprotonation of the active site histidine can act as a switch for product release. This study connects thermodynamics with the structures of the pre- and postcatalytic complex, clarifying previous experimental results and emphasizing the design of 18-ComC-based peptide inhibitors for PEP in S. mutans.