Abstract The latest wave SARS-CoV-2 Omicron variants displayed a growth advantage and the increased viral fitness through convergent evolution of functional hotspots that work synchronously to balance fitness requirements for productive receptor binding and efficient immune evasion. In this study, we combined AlphaFold2-based structural modeling approaches with all-atom MD simulations and mutational profiling of binding energetics and stability for prediction and comprehensive analysis of the structure, dynamics, and binding of the SARS-CoV-2 Omicron BA.2.86 spike variant with ACE2 host receptor and distinct classes of antibodies. We adapted several AlphaFold2 approaches to predict both structure and conformational ensembles of the Omicron BA.2.86 spike protein in the complex with the host receptor. The results showed that AlphaFold2-predicted conformational ensemble of the BA.2.86 spike protein complex can accurately capture the main dynamics signatures obtained from microscond molecular dynamics simulations. The ensemble-based dynamic mutational scanning of the receptor binding domain residues in the BA.2 and BA.2.86 spike complexes with ACE2 dissected the role of the BA.2 and BA.2.86 backgrounds in modulating binding free energy changes revealing a group of conserved hydrophobic hotspots and critical variant-specific contributions of the BA.2.86 mutational sites R403K, F486P and R493Q. To examine immune evasion properties of BA.2.86 in atomistic detail, we performed large scale structure-based mutational profiling of the S protein binding interfaces with distinct classes of antibodies that displayed significantly reduced neutralization against BA.2.86 variant. The results quantified specific function of the BA.2.86 mutations to ensure broad resistance against different classes of RBD antibodies. This study revealed the molecular basis of compensatory functional effects of the binding hotspots, showing that BA.2.86 lineage may have primarily evolved to improve immune escape while modulating binding affinity with ACE2 through cooperative effect of R403K, F486P and R493Q mutations. The study supports a hypothesis that the impact of the increased ACE2 binding affinity on viral fitness is more universal and is mediated through cross-talk between convergent mutational hotspots, while the effect of immune evasion could be more variant-dependent.

Accurate Characterization of Conformational Ensembles and Binding Mechanisms of the SARS-CoV-2 Omicron BA.2 and BA.2.86 Spike Protein with the Host Receptor and Distinct Classes of Antibodies Using AlphaFold2-Augmented Integrative Computational Modeling