AbstractRecently, a cluster of several newly occurring mutations on Omicron, which is currently the dominant SARS-CoV-2 variant, are found at the (mechanically) stable β-core region of spike protein’s receptor-binding domain (RBD), where mutation rarely happened before. Notably, the binding of SARS-CoV-2 to human receptor ACE2 via RBD happens in a dynamic airway environment, where mechanical force caused by coughing or sneezing occurs and applies to the proteins. Thus, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to measure the stability of RBDs and found that the unfolding force of Omicron RBD increased by 20% compared with the wild-type. Molecular dynamics simulations revealed that Omicron RBD showed more hydrogen bonds in the β-core region due to the closing of the α-helical motif caused primarily by the S373P mutation, which was further confirmed by the experiment. This work reveals the stabilizing effect of the S373P mutation and suggests mechanical stability becomes another important factor in SARS-CoV-2 mutation selection.