Energy Landscape of the SARS-CoV-2 Reveals Extensive Conformational Heterogeneity

Cryo-electron microscopy (cryo-EM) has produced a number of structural models of the SARS-CoV-2 spike, already prompting biomedical outcomes. However, these reported models and their associated electrostatic potential maps represent an unknown admixture of conformations stemming from the underlying energy landscape of the spike protein. As for any protein, some of the spikes conformational motions are expected to be biophysically relevant, but cannot be interpreted only by static models. Using experimental cryo-EM images, we present the energy landscape of the spike protein conformations, and identify molecular rearrangements along the most-likely conformational path in the vicinity of the open (so called 1RBD-up) state. The resulting global and local atomic refinements reveal larger movements than those expected by comparing the reported 1RBD-up and 1RBD-down cryo-EM models. Here we report greater degrees of "openness" in global conformations of the 1RBD-up state, not revealed in the single-model interpretations of the density maps, together with conformations that overlap with the reported models. We discover how the glycan shield contributes to the stability of these conformations along the minimum free-energy pathway. A local analysis of seven key binding pockets reveals that six out them, including those for engaging ACE2, therapeutic mini-proteins, linoleic acid, two different kinds of antibodies, and protein-glycan interaction sites, switch conformations between their known apo- and holo-conformations, even when the global spike conformation is 1RBD-up. This is reminiscent of a conformational pre-equilibrium. We found only one binding pocket, namely antibody AB-C135 to remain closed along the entire minimum free energy path, suggesting an induced fit mechanism for this enzyme.

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

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 in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 s of weighted ensemble (WE) 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-EM 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 achieves a new high-water mark for ensemble pathway simulations and offers a foundation for understanding the fundamental mechanisms of SARS-CoV-2 viral entry and infection.