Supramolecular assembly and membrane anchoring alter variant-induced SARS-CoV-2 Spike conformational dynamics

Viral surface glycoproteins function within crowded, membrane-anchored supramolecular assemblies, yet their dynamics are often inferred from soluble, engineered ectodomains. For SARS-CoV-2 Spike (S), this distinction is especially important because variant mutations evolved in the context of intact virions, not engineered trimers. Here, we combined cyclic ion mobility mass spectrometry (CIM-MS) for hydrogen deuterium exchange mass spectrometry (HDXMS) with computational weighted ensemble molecular dynamic (WE-MD) simulations to compare the conformational effects of SARS-CoV-2 mutations in two S protein constructs: soluble recombinant 2P S and full-length S displayed in virus-like particles (VLPs). Our results reveal that membrane anchoring and virion-like assembly substantially dampen global dynamic changes produced by variant mutations. In VLP S, D614G induced decreased dynamics across interprotomer contacts, whereas Omicron BA.1 mutations systematically reversed this decrease in dynamics in the S1 domain while preserving decreased dynamics near the fusion machinery. These effects are more spatially constrained than those observed in recombinant 2P S. Comparison of the 2 constructs further reveals that 2P substitutions alter native interprotomer communication. Recombinant S shows a predominantly fast-exchanging population in the receptor binding domain (RBD) hinge region, consistent with disruption of hydrogen-bonding and salt bridge networks that couple RBD motion to the trimer core. Thus, recombinant S captures important structural features of S without recapitulating the native conformational ensemble presented on virions. Together, these results show that the supramolecular context of S reshapes the conformational dynamics of variant evolution with direct implications for epitope presentation, antibody recognition, and interpretation of recombinant S structural studies.