A glycan gate controls opening of the SARS-CoV-2 spike protein

Sztain, Terra; Ahn, Surl-Hee; Bogetti, Anthony T; Casalino, Lorenzo; Goldsmith, Jory A; Seitz, Evan; McCool, Ryan S; Kearns, Fiona L; Acosta-Reyes, Francisco; Maji, Suvrajit; Mashayekhi, Ghoncheh; McCammon, J Andrew; Ourmazd, Abbas; Frank, Joachim; McLellan, Jason S; Chong, Lillian T; Amaro, Rommie E

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded 'down' to an exposed 'up' state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the 'up' and 'down' states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways. Together with ManifoldEM analysis of cryo-electron microscopy data and biolayer interferometry experiments, we reveal a gating role for the N-glycan at position N343, which facilitates RBD opening. Residues D405, R408 and D427 also participate. The atomic-level characterization of the glycosylated spike activation mechanism provided herein represents a landmark study for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.