Structural insights into subunit exchange inhibition and ligand‐induced dimerization of SARS ‐ CoV ‐2 main protease

The SARS‐CoV‐2 main protease (M pro ) is an essential homodimeric cysteine protease that mediates viral polyprotein processing and represents a primary therapeutic target for COVID‐19 treatment. While peptidomimetic inhibitors such as nirmatrelvir have demonstrated clinical efficacy, their effects on M pro quaternary structure dynamics remain poorly understood. Emerging evidence suggests these compounds may paradoxically promote dimerization, a phenomenon termed ligand‐induced dimerization (LID), which could enhance catalytic activity at low inhibitor concentrations. Here, we employed an integrated mass spectrometry platform combining metabolic 15 N labeling with native mass spectrometry (nMS), hydrogen‐deuterium exchange mass spectrometry (HDX‐MS), and molecular dynamics (MD) simulations to elucidate the dynamic nature of M pro dimerization and its modulation by peptidomimetic inhibitors. We demonstrate that apo‐M pro exists in a dynamic monomer–dimer equilibrium characterized by rapid subunit exchange. Remarkably, peptidomimetic inhibitors, including nirmatrelvir, GC376, and MPI8, kinetically trap the dimeric state, suppressing subunit exchange and stabilizing the dimeric form of M pro . Region‐resolved HDX‐MS revealed that inhibitor binding induces widespread conformational stabilization extending beyond the active site to the N‐finger (residues 1–17) and dimer interface (residues 291–302). A notable transition of exchange mode from bimodal (EX1) to unimodal (EX2) in the N‐finger region provided a sensitive structural marker of ligand‐induced dimerization. Molecular dynamics simulations confirmed that the observed stabilization arises primarily from dimer formation rather than direct ligand contacts. These findings establish a mechanistic basis for inhibitor‐stabilized dimerization, identify the N‐finger as a dynamic reporter of quaternary structure transitions, and demonstrate the power of integrated mass spectrometry for dissecting complex protein assembly dynamics with therapeutic implications for antiviral drug development.