A hierarchically organized communication network underlies kinetic allostery in SARS-CoV-2 M^pro

The SARS-CoV-2 main protease (M^pro) is an essential viral enzyme whose catalytic activity strictly depends on dimerization and inter-protomer functional communication. While hundreds of symmetrical ligand-free or fully occupied structures are available, the structural and dynamical mechanisms governing asymmetric long-range communication remain elusive, hampering the rational development of non-competitive, allosteric inhibitors. Here, we leverage unconventional, asymmetrical crystal structures captured in a singly occupied, peptide-bound state to uncover the structural baseline of M^pro kinetic allostery. By integrating comparative crystallographic analysis with anisotropic network model calculations, perturbation response scanning, network centrality, and frustration profiling, we demonstrate that substrate binding to a single subunit induces minimal structural rearrangements. This indicates that inter-protomer coupling is not mediated by large conformational changes but rather operates through a highly specialized dynamical scaffold. We resolve this scaffold into a hierarchically organized allosteric network composed of interfacial driver modules, internal relay hubs, and peripheral receiver-like regions. Specifically, core residues 1-10 and 123-128 emerge as key communication gates, while domain III acts as a distinct secondary modulatory branch. By mapping the exact topological routing of allosteric signal propagation, this study moves beyond traditional static descriptions, providing a rigorous structural blueprint of M^pro intrinsic regulation. These conserved, functionally central hotspots provide an ideal molecular map not only for the rational design of next-generation allosteric antivirals designed to disrupt inter-subunit communication, but also for assessing the structural impacts of emerging M^pro variants and predicting resistance mutations outside the active site.