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We integrate evolutionary predictions based on the neutral theory of molecular evolution with protein dynamics to generate mechanistic insight into the molecular adaptations of the SARS-COV-2 spike (S) protein. With this approach, we first identified candidate adaptive polymorphisms (CAPs) of the SARS-CoV-2 S protein and assessed the impact of these CAPs through dynamics analysis. Not only have we found that CAPs frequently overlap with well-known functional sites, but also, using several different dynamics-based metrics, we reveal the critical allosteric interplay between SARS-CoV-2 CAPs and the S protein binding sites with the human ACE2 (hACE2) protein. CAPs interact far differently with the hACE2 binding site residues in the open conformation of the S protein compared to the closed form. In particular, the CAP sites control the dynamics of binding residues in the open state, suggesting an allosteric control of hACE2 binding. We also explored the characteristic mutations of different SARS-CoV-2 strains to find dynamic hallmarks and potential effects of future mutations. Our analyses reveal that Delta strain-specific variants have non-additive (i.e., epistatic) interactions with CAP sites, whereas the less pathogenic Omicron strains have mostly additive mutations. Finally, our dynamics-based analysis suggests that the novel mutations observed in the Omicron strain epistatically interact with the CAP sites to help escape antibody binding.

We develop integrated co-evolution and dynamic coupling (ICDC) approach to identify, mutate, and assess distal sites to modulate function. We validate the approach first by analyzing the existing mutational fitness data of TEM-1 β-lactamase and show that allosteric positions co-evolved and dynamically coupled with the active site significantly modulate function. We further apply ICDC approach to identify positions and their mutations that can modulate binding affinity in a lectin, cyanovirin-N (CV-N), that selectively binds to dimannose, and predict binding energies of its variants through Adaptive BP-Dock. Computational and experimental analyses reveal that binding enhancing mutants identified by ICDC impact the dynamics of the binding pocket, and show that rigidification of the binding residues compensates for the entropic cost of binding. This work suggests a mechanism by which distal mutations modulate function through dynamic allostery and provides a blueprint to identify candidates for mutagenesis in order to optimize protein function.