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

Abstract

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.

Figure 1

Glycosylated spike RBD “down” and “open” conformations. (A) The SARS-CoV-2 spike head (gray) with glycans (dark blue) as simulated, with the stalk domain and membrane (not simulated here, but shown in transparent for completeness). RBD shown in cyan, receptor binding motif (RBM) in pink. Side view of the “down” (shielded, B) and “open” (exposed, C) RBD. Top view of the closed (shielded, D) and “open” (exposed, E) RBM. Composite image of glycans (dark blue lines) shows many overlapping snapshots of the glycans over the microsecond simulations.

Figure 2

Atomically detailed pathways of spike opening. (A-E) Snapshot configurations along the opening pathway with chain A shown in cyan, chain B in gray, and chain C in pink and the glycan at position N343 is shown in magenta. (F) Scatter plot of data from the 310 continuous pathways with the Cα RMSD of the RBD from the 6VSB “up” state plotted against the RBD —core distance. Data points are colored based on % RBD solvent accessible surface area compared to the RBD “down” state 6VXX. Location of snapshots shown in A-E are labeled. (G) Primary regions of spike defined for tracking progress of the opening transition. The spike core is composed of three central helices per trimer, colored according to chain as in (A-E). The RBD contains a structured pair of antiparallel beta sheets and an overlay of snapshots from a continuous WE simulation are shown colored along a spectrum resembling the palette in (F). Overlayed cryoEM structures are highlighted and labeled including the initial RBD “down” state, 6VXX, the target RBD “up” state and the ACE2 bound “open” state, 7A95.

Figure 3

Glycan gating by N343. (A-D) Snapshot configurations along the opening pathway with chain A shown in cyan, chain B in gray, chain C in pink, and the glycan at position N343 is shown in magenta. (A) RBD A in the “down” conformation is shielded by the glycan at position N343 of the adjacent RBD B. (B-D) The N343 glycan intercalates between and underneath the residues F490, Y489, F456, F457 to push the RBD up and open (D).

Figure 4

ACE2 binding is reduced by mutation of N343 glycosylation site and key salt bridge residues. (A) Biolayer interferometry sensorgrams of HexaPro spike variants binding to ACE2. For clarity, only the traces from the first replicate are shown. (B) Graph of binding response for BLI data collected in triplicate with error bars representing the standard deviation from the mean.

Figure 5

Salt bridges and hydrogen bonds along the opening pathway. (A-D) salt bridge or hydrogen bond contacts made between RBD A, shown in blue, with RBD B, shown in gray, or RBD C shown in pink within the “down”, transient, “up”, and “open” conformations, respectively. (E) Histogram showing the frequency at which residues from (A-D) are within 3.5 Å of each other relative to RBD — core distance. Frequencies are normalized to 1.