Structural and functional impact of MProG48Y/ΔP168 mutations in SARS-CoV-2 main protease exhibiting resistance to potent inhibitors

TL;DR

This study investigates how specific mutations in the SARS-CoV-2 main protease affect its structure, function, and resistance to inhibitors.

Contribution

The study reveals how MProG48Y/ΔP168 mutations alter protease dynamics and reduce inhibitor affinity while maintaining enzymatic activity.

Findings

MProG48Y/ΔP168 retains efficient autoprocessing and catalytic activity similar to the wild type.

The mutant shows a 3- to 5-fold reduced affinity for nirmatrelvir and ensitrelvir but unchanged affinity for GC373.

Structural and dynamic changes in the active site suggest compensatory adjustments in the mutant protease.

Abstract

Mutations in the main protease (MPro) can alter its enzymatic function and influence drug susceptibility, impacting SARS-CoV-2 maturation and propagation. In this study we examine the effects of G48Y and ΔP168 mutation/deletion, located in the substrate-binding subsites S2 and S4, on dimer stability, enzymatic activity, inhibitor binding, and active site dynamics. Despite these mutations, MProG48Y/ΔP168 retains efficient N- terminal autoprocessing and catalytic activity comparable to the wild type (MProWT). Structural analysis reveals an open active site conformation, increased flexibility in the S2 helix, S5 loop, and helical domain, and a ∼40-fold reduction in dimer dissociation constant (Kdimer), despite an 8°C decrease in thermal sensitivity to unfolding. Thermodynamic binding studies indicate a 3-fold and 5-fold reduced affinity for nirmatrelvir and ensitrelvir, respectively, while GC373 binding remains unchanged relative to MProWT. Structural comparisons highlighted an open S2 helix and S5 loop conformation in both apo and inhibitor-bound states, suggesting mutational effects are compensated through dynamic adjustments that modulate autoprocessing, dimer stability, and inhibitor interactions. Comparison of MProG48Y/ΔP168 and MProWT based on B-factor analysis and MD simulations corroborates with enhanced dynamics of the active site and of the entire helical region in the mutant relative to the wild-type enzyme.