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 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 200 μs 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 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.

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…In another recent study, RBD N-glycosylation site mutants were expressed in HEK293 cells without any obvious effects on expression levels, but decreased ACE2 binding upon removal of the N-glycans at both sites was reported ( Azad et al, 2021 ). In agreement with the later finding, the glycan at position N343 may play an important role for the opening mechanism of the spike and stabilization of RBD in the up state that is required for ACE2 binding and cell entry ( Sztain et al, 2021 ). In plants, neither the individual RBD-215 N-glycosylation mutants nor the RBD-215 variant lacking both N-glycosylation sites (RBD-215-12Q) could be produced as soluble proteins, suggesting that both N-glycans contribute to proper folding.…”

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

“…Nevertheless, the unique RBD binding mode of Nb17 and its ease of bioengineering may facilitate the development of robust biosensors to detect specific conformations of the spike. Currently, this process can only be inferred computationally [36][37][38][39] or by single-molecule techniques which require extensive bioengineering to label the spike with fluorescence dyes 17 . Finally, since Nb17 binds extremely tightly to the RBD-up conformations, potentially, it could be used as a novel Nb "adjuvant" for vaccines to facilitate exposure of conserved yet cryptic RBD epitopes that could help elicit broadly neutralizing activities insensitive to the evolving variants.…”

Structure-function relations of the SARS-CoV-2 spike protein and impact of mutations in the variants of concern

“…Deux autres catégories d'anticorps ont été identifiées dans lesquelles l'épitope est en dehors du patch ACE2 : les anticorps de classe 3 ciblent une surface exposée, tandis que l'épitope de la classe 4 est enfoui dans le spicule fermé et nécessite au moins deux RBD debout pour la liaison [109][110][111][112]. Les anticorps de classe 4 sont parmi Il est à noter que les simulations par dynamique moléculaire suggèrent que le glycane N343 joue un rôle de barrière dans le basculement du RBD vers la conformation debout [96]. Le panneau inférieur montre une vue de dessus sans mettre en évidence le patch ACE2.…”

“…The glycan chains interacting with the RBD on the closed spike are shown as sticks colored according to atom type: nitrogen blue, oxygen red, and carbon atoms light grey for the N343 glycan attached to the represented protomer, and yellow for those emanating from adjacent protomers (N149 in a neighboring NTD and N343 in the adjacent RBD), showing that they mask part of the surface not buried by protein atoms as displayed in panel A. Of note, molecular dynamics simulations suggest a gating role of the N343 glycan to allow swinging of the RBD to the Up conformation[96]. The lower panel shows a top view without highlighting the ACE2 patch.…”

“…(A, B) La surface du RBD colorée selon son accessibilité au solvant dans la conformation fermée du spicule (A) et selon la conservation des acides aminés (B) comme indiqué dans la barre colorée. (C) La surface de liaison de ACE2 a été superposée en jaune sur la même vue qu'en A. Les chaînes de glycanes interagissant avec le RBD sur le spicule fermé sont représentées sous forme de bâtonnets colorés selon le type d'atome : bleu pour l'azote, rouge pour l'oxygène et gris clair pour les atomes de carbone du glycan N343 attaché au protomère représenté, et jaune pour ceux émanant des protomères adjacents (N149 dans un NTD voisin et N343 dans le RBD adjacent), montrant qu'ils masquent une partie de la surface non enfouie dans le trimère fermé par des atomes de la chaine polypeptidique comme indiqué dans le panneau A. Il est à noter que les simulations par dynamique moléculaire suggèrent que le glycane N343 joue un rôle de barrière dans le basculement du RBD vers la conformation debout[96]. Le panneau inférieur montre une vue de dessus sans mettre en évidence le patch ACE2.…”