Interfacial subregions of SARS-CoV-2 spike RBD to hACE2 affect intermolecular affinity by their distinct roles played in association and dissociation kinetics.

SARS-CoV-2's rapid global transmission depends on spike RBD's strong affinity to hACE2. In the context of binding hot spots well defined, the work investigated how interfacial subregions of SARS-CoV-2 spike RBD to hACE2 affect intermolecular affinity and their potential distinct roles involved in association and dissociation kinetics due to their local structural characteristics. Three spatially consecutive subregions of SARS-CoV-2 RBD were structurally partitioned across RBD's receptor binding motif (RBM). Their impacts on binding affinity and kinetics were differentiated through a comprehensive SPR measurement of hACE2 binding by chimeric swap mutants of respective subdomains from SARS-CoV-2 VOCs & phylogenetically close sarbecoviruses, and further compared with those of included single mutations across RBM and around the RBD core. The data supports that the intermediate interfacial subregion of RBD involving key residue at 417 is the rate-limiting effector of association kinetics and the subregion encompassing residues at 501/498/449 is the key binding energy contributor dictating dissociation kinetics, both of which relate to SARS-CoV-2's adaptive mutational evolution and host tropism closely. The kinetic data and structural analysis of local mutations' impact on spike RBD's binding and thermal stability provide a new perspective in evaluating SARS-CoV-2 evolution and other sarbecoviruses' evolvable binding to hACE2. The inherent binding mode offers direct clues of valid epitope in designing new antibodies that the coronavirus can't elude.