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

Abstract

The SARS-CoV-2 main protease (Mpro) 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 Mpro 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 15N labeling with native mass spectrometry (nMS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics (MD) simulations to elucidate the dynamic nature of Mpro dimerization and its modulation by peptidomimetic inhibitors. We demonstrate that apo-Mpro 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 Mpro. 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.

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