Allostery Links hACE2 Binding, Pan-variant Neutralization and Helical Extension in the SARS-CoV-2 Spike Protein

The SARS-CoV-2 spike protein is highly antigenic, with epitopes in three distinct regions of the receptor binding domain (RBD) alone that have known mechanisms of neutralization. In previous work, we predicted a fourth RBD epitope based on allosteric conformational perturbations measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS) upon complexation with the canonical spike protein target, human angiotensin-converting enzyme 2 (hACE2). We subsequently identified a pan-neutralizing antibody (ICO-hu104) with the predicted epitope, however, as the epitope was somewhat distant from the hACE2 binding interface, and our previous work limited to the spike RBD, the neutralization mechanism was unclear. Using HDX-MS, we investigated the binding of ICO-hu104 to the full-length SARS-CoV-2 spike protein from Wuhan, Delta and Omicron variants. We demonstrate that binding of ICO-hu104 results in an increase in deuterium uptake in the distant HR1 domain in latter variants, which in a biological context could be indicative of destabilisation of the helices within this region, promoting premature S1 shedding or failure of helical extension during S2-mediated fusion. This is supported by our computational modelling, highlighting propagation of allosteric effects to the S2 coiled-coil region upon rigidification of the ICO-hu104 epitope. Collectively, this work demonstrates an alternative neutralization mechanism for ICO-hu104 which is distinct from its first-generation predecessors and thus opens alternative avenues targeting non-RBD epitopes through allosteric perturbations.