Mechanochemical Decoupling of ATP Hydrolysis and RNA Translocation in SARS-CoV-2 nsp13 by the L405D Mutation

Abstract SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved helicase that couples ATP hydrolysis to RNA translocation through long-range allosteric communication between its ATPase and RNA-binding domains. In prior work, we identified L405 as a key regulator of interdomain motions and proposed that the L405D mutation would disrupt this coupling by perturbing conformational transitions required for translocation [J. Phys. Chem. B2024, v128, 492–503. ]. Subsequent experiments confirmed that L405D attenuates helicase activity and ATPase activity, implicating the potential formation of a new salt bridge, D405-R560, for both effects [ J. Biol. Chem.2026, v302, 111198.]. Here, we provide a data-driven explanation for both effects by combining Gaussian accelerated molecular dynamics (GaMD) simulations with Shape-GMM clustering and linear discriminant analysis. Whereas wild-type nsp13 exhibits both conformational selection and induction, L405D collapses the conformational landscape to operate predominantly through selection, with ATP-induced structural transitions observed in the wild-type ensemble no longer sampled by the mutant. This loss of induction likely impairs ATP turnover while simultaneously disrupting coordinated motif–RNA interactions required for inchworm translocation. These findings demonstrate how mutation-induced reshaping of conformational ensembles can modulate access to reaction-competent states, providing a general framework for understanding how targeted mutations disrupt catalytic function through allosteric ensemble remodeling in motor proteins.