Comparative molecular dynamics mapping of metallocarboxypeptidase-peptide interfaces reveals potential hotspots that inspire novel inhibitor design.

Metallocarboxypeptidases (MCPs) are critical zinc-containing exopeptidases and promising therapeutic targets for conditions ranging from thromboembolic disorders to cancer. Despite their importance, the dynamic determinants governing subfamily-specific peptide recognition remain incompletely understood. In this study, we performed 200 ns molecular dynamics simulations on five distinct A/B MCP-peptide complexes and integrated contact analysis and per-residue energy decomposition (MM/PBSA) to systematically map their interaction profiles. We identified a core set of Grade A and B conserved hotspots-specifically Arg71, Arg127, Glu163, Thr164, and Tyr248-that mediate persistent interactions across the subfamilies. Based on their energetic and geometric profiles, we categorized these residues into three functional classes. Class I (functional hotspots): Such as Arg71 and Arg127, which act as near-permanent "anchoring hubs" with contact occupancies exceeding 90% and dominant negative binding free energies. Class II (topological hotspots): Such as Glu163, which maintains the structural integrity of the active site cleft. Class III (auxiliary energetic contributors): Which provide additional stabilization through transient or non-polar contacts. Furthermore, we identified complex-specific residues, such as Glu270 in hCPB1, that serve as selectivity filters. Collectively, this study highlights the key enzyme residues that mediate persistent interactions at the interface and provides molecular insights that may guide the rational design of novel metallocarboxypeptidase inhibitors.