BPS2026 – Covariance-based MD simulation analysis pinpoints nanobody attraction and repulsion sites in SARS-CoV-2 Omicron spike protein

The heavily mutated receptor-binding domain (RBD) of the SARS-CoV-2 Omicron spike protein poses a major challenge to neutralizing antibodies and nanobodies, yet the molecular basis of disrupted binding is not yet fully elucidated. Here, we present an all-atom molecular dynamics (MD) investigation of fourteen nanobodies (H11-H4, H11-D4, RE5D06, WNB2, MR17, Huo-H3, SB15, VHH-E, Ty1, NM1230, SB23, SB14, NB21, and SB45) bound to the Omicron RBD, altogether encompassing multiple microseconds of MD sampling. To map the determinants of binding, we combined MD trajectories with a covariance-based method to identify stabilizing and destabilizing interactions at nanobody-RBD interfaces, focusing on complementarity-determining regions (CDRs) that mediate binding and recognition. This integrated approach revealed residue-specific hydrogen bonds, salt bridges, and hydrophobic contacts that stabilize binding, as well as repulsive electrostatic and steric clashes introduced by Omicron substitutions. Across nanobody ensembles, E484A emerged as a major contributor to electrostatic repulsion, destabilizing multiple interfaces and correlating with reduced binding stability. While each nanobody displayed distinct poses and interaction networks, common RBD “hotspots” were engaged by multiple nanobodies, while unique contacts explained selective sensitivity to Omicron mutations. Correlation analyses further showed that repulsive substitutions altered long-range coupling across nanobody-RBD complexes, disrupting stabilizing networks. Together, these results establish an integrated MD-covariance workflow that quantifies how CDR sequence variation and viral mutations shape binding energetics. By pinpointing stabilizing networks and mutation-induced clash sites, our study provides a mechanistic framework for rational engineering of next-generation nanobodies with sustained potency against SARS-CoV-2 evolution.