A hierarchically organized communication network underlies kinetic allostery in SARS-CoV-2 Mpro

: The SARS-CoV-2 main protease (Mpro) 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 Mpro 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 Mpro 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 Mpro variants and predicting resistance mutations outside the active site.