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

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 translocations required for translocation [J. Phys. Chem. B 2024 v128 492–503]. Subsequent experiments confirmed that L405D attenuates helicase activity while largely preserving ATPase activity, implicating a breakdown in ATP-to-RNA coupling [J. Biol. Chem. 2026 v302 111198]. Here, we provide a data-driven explanation for this decoupling 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, eliminating ATP-induced structural transitions required for efficient catalytic cycling. This loss of induction traps the ATP-binding pocket in a mid-open conformation, impairing product release and reducing ATP turnover, while simultaneously disrupting coordinated motif–RNA interactions required for inchworm translocation. These findings establish that 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.