Conformational dynamics of SARS-CoV-2 spike on a membrane reveals the allosteric effects of furin cleavage and the D614G mutation

By combining hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) with the ability of enveloped virus–like particles (eVLPs) to display full-length native-like severe acute respiratory syndrome coronavirus 2 spike protein, we have determined the energetic and conformational effects of both the membrane environment and unique sequence features that are considered incompatible with soluble protein constructs. We find that eVLP-displayed spike can sample the open-interface trimer conformation observed in soluble constructs of spike, including sequences from engineered vaccine constructs and native viral sequences inaccessible to studies on soluble constructs. Moreover, the D614G mutation, which arose early in the pandemic, favors the canonical “closed-interface” prefusion conformation, potentially mitigating premature S1 shedding in the presence of a cleaved furin site and providing an evolutionary advantage to the virus. Furin cleavage at the S1/S2 boundary allosterically increases the flexibility of the S2′ site, which may facilitate increased TMPRSS2 processing, enhancing viral infectivity. The use of eVLPs in HDX-MS studies provides a powerful platform for studying viral and membrane proteins in near-native environments.