Differential Impact of Substrate Peptides on Interdomain Interactions in Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease

Severe acute respiratory syndrome coronavirus 2 main protease (M pro ) is essential for viral replication by cleaving polyproteins pp1a and pp1ab at 11 sites into functional proteins and remains a major pharmacological target. Although its structure and catalytic mechanism are well characterized, how different substrate peptides dynamically interact with and influence M pro remains incompletely understood. To study these substrate-peptide-specific effects on M pro structural dynamics, we used molecular dynamics (MD) simulations of the M pro dimer bound to its cognate substrate peptides individually and complemented by analyses revealing substrate-peptide-specific structural changes in M pro . Specifically, we generated structural models of all M pro –substrate peptide complexes and performed all-atom, explicit solvent MD simulations. MD trajectory and SHapley Additive exPlanations (SHAP) analyses indicated that substrate peptides modulate M pro dynamics in a substrate-specific manner, predominantly affecting the T45 to M49 and R188 to Q192 residues in the catalytic site. Importantly, this influence does not arise from a single conserved substrate position but from distinct residues across different substrate peptides, highlighting dynamic and context-dependent coupling. Furthermore, hydrogen bond (H-bond) interaction analysis showed substrate-peptide-specific differences in interdomain H-bond interaction between domains I and II. Together, these findings demonstrate that M pro does not respond uniformly to substrate peptide binding; rather, each substrate peptide uniquely reshapes the flexibility of catalytic site residues and interdomain coupling, with potential implications for substrate recognition and inhibitor design.