Covariance-Based MD Simulation Analysis Pinpoints Nanobody Attraction and Repulsion Sites on SARS-CoV-2 Omicron Spike Protein

Omicron’s heavily mutated SARS-CoV-2 spike receptor-binding domain (RBD) enables broad escape from neutralizing antibodies and nanobodies (Nbs), yet the atomistic basis of epitope-dependent Nb loss remains unclear. We performed >8 μs of all-atom molecular dynamics (MD) simulations of 13 Nbs (H11-D4, H11-H4, Huo-H3, MR17, NB21, NM1230, RE5D06, SB14, SB23, SB45, Ty1, VHH-E, WNB2) bound to the Omicron RBD to characterize binding-pose stability and interfacial dynamics. Principal-component analysis of MD trajectories yields binding-pose free-energy landscapes: most Nbs occupy single dominant basins yet often deviate from WTbound poses (orientation shifts), whereas NB21 and SB14 show pose plasticity. Using our covariance-matrix–based interaction-mapping method, we map stabilizing versus destabilizing residue–residue couplings and classify the underlying attractive interactions (salt bridges, hydrogen bonds, hydrophobic contacts) and repulsive interactions (like-charge repulsion or polarity mismatches). Attractive contacts converge on recurrent hydrophobic “anchor” patches on the receptor-binding motif (RBM) (∼V445-G456 and ∼F490-Y501) alongside binder-specific, complementarity-determining-region (CDR)-shaped interactions. Omicron substitutions rewire contact networks, shift binding orientations, and can introduce unfavorable repulsion, directly at mutation sites (e.g., E484A) or indirectly via mutation-driven reorientation, weakening binding. Mutation-induced repulsion reshapes correlated interfacial motions and reduces interfacial stability, providing a mechanistic basis for diminished binding robustness of multiple Nbs that were effective against WT and/or earlier variants. Low-speed steered MD pulling (AFM comparable) of ACE2 and selected Nbs shows lower unbinding work for Nbs than ACE2 (∼16–42 vs ∼45 kcal/mol), with NM1230 and SB23 most mechanically robust. These Nb–RBD interaction fingerprints pinpoint epitope-specific determinants and mutation-induced clash sites, providing a blueprint for structure-guided CDR engineering of Nbs with sustained potency against ongoing SARS-CoV-2 evolution.