Covariance-Based MD Simulation Analysis Pinpoints Nanobody Attraction and Repulsion Sites on SARS-CoV-2 Omicron Spike Protein
Mutations in the SARS-CoV-2 spike receptor-binding domain (RBD) of the Omicron variant enable broad escape from neutralizing antibodies (Abs) and nanobodies (Nbs), yet the atomistic basis of epitope-dependent loss of Nb binding remains unclear. We performed all-atom molecular dynamics (MD) simulations of 13 Nbs bound to the Omicron RBD to characterize their binding modes, binding-pose stability, and interfacial dynamics. Analysis of the trajectories and corresponding free-energy landscapes revealed that most Nbs occupied a single dominant basin but showed varying degrees of within-basin heterogeneity and often exhibited orientation shifts, whereas two Nbs (Nb21 and Sb14) showed pronounced pose plasticity and dissociated from the RBD. For the remaining 11 Nbs, covariance-matrix analysis mapped stabilizing and destabilizing residue couplings, revealing recurrent hydrophobic anchor patches within the receptor-binding motif and Nb-specific interaction patterns reflecting differences in the sequences and chemistries of their complementarity-determining regions. Omicron substitutions rewired interaction networks, shifted Nb binding orientations relative to the corresponding experimentally determined reference structures (obtained with the ancestral WT RBD for all Nbs except H3), and introduced unfavorable repulsion, either directly at mutation sites or indirectly via mutation-driven reorientation, thereby reducing interfacial stability. Low-speed steered MD unbinding simulations of the 11 Nb-RBD complexes and the ACE2-RBD complex, performed at a pulling velocity within the range used in high-speed AFM-based force spectroscopy, yielded lower mean unbinding work for all 11 Nbs than for ACE2. These simulations also revealed heterogeneous, stepwise Nb unbinding pathways in which receptor-binding ridge (RBR)-mediated anchoring maintained partial Nb attachment through newly formed interactions and stabilized transient intermediate binding poses. Together, these Nb-RBD interaction fingerprints and pathway-resolved unbinding simulations pinpoint epitope-specific determinants and mutation-induced clash sites, providing a mechanistic basis for diminished Nb binding.