Despite an unprecedented global gain in knowledge since the emergence of SARS-CoV-2, almost all mechanistic knowledge related to the molecular and cellular details of viral replication, pathology and virulence has been generated using early prototypic isolates of SARS-CoV-2. Here, using atomic force microscopy and molecular dynamics, we investigated how these mutations quantitatively affected the kinetic, thermodynamic and structural properties of RBD—ACE2 complex formation. We observed for several variants of concern a significant increase in the RBD—ACE2 complex stability. While the N501Y and E484Q mutations are particularly important for the greater stability, the N501Y mutation is unlikely to significantly affect antibody neutralization. This work provides unprecedented atomistic detail on the binding of SARS-CoV-2 variants and provides insight into the impact of viral mutations on infection-induced immunity.

Unveiling the nanomechanical stability of the novel coronavirus (SARS-CoV-2) spike protein

We used single-molecule AFM force spectroscopy (AFM-SMFS) in combination with click chemistry to mechanically dissociate anticalin, a non-antibody protein binding scaffold, from its target (CTLA-4), by pulling from eight different anchor residues. We found that pulling on the anticalin from residue 60 or 87 resulted in significantly higher rupture forces and a decrease in k off by 2–3 orders of magnitude over a force range of 50–200 pN. Five of the six internal anchor points gave rise to complexes significantly more stable than N- or C-terminal anchor points, rupturing at up to 250 pN at loading rates of 0.1–10 nN s–1. Anisotropic network modeling and molecular dynamics simulations helped to explain the geometric dependency of mechanostability. These results demonstrate that optimization of attachment residue position on therapeutic binding scaffolds can provide large improvements in binding strength, allowing for mechanical affinity maturation under shear stress without mutation of binding interface residues.