Fine-tuning the Spike: Role of the nature and topology of the glycan shield in the structure and dynamics of SARS-CoV-2 S

Harbison, Aoife M.; Fogarty, Carl A.; Phung, Toan K.; Satheesan, Akash; Schulz, Benjamin L.; Fadda, Elisa

The SARS-CoV-2 spike (S) is a type I fusion glycoprotein, responsible for initiating the infection leading to COVID19. As a feature unique of SARS-CoV-2, the thick glycan shield covering the S protein is not only essential for hiding the virus from immune detection, but it also plays multiple functional roles, stabilizing the S prefusion “open” conformation, which is competent for binding the ACE2 primary receptor, and gating the open-to-close transitions. This newly discovered functions of the glycan shield suggest the evolution of its sites of glycosylation is potentially intertwined with the evolution of the overall protein sequence to affect optimal activity. Furthermore, recent studies indicate that the occupancy and structures of SARS-CoV-2 S glycosylation depends not only on the host-cell, but also on the structural stability of the prefusion trimer; a point that raises important questions about the relative binding competence of different glycoforms. In this work we use multi-microsecond molecular dynamics simulations to characterize the structure and dynamics of different SARS-CoV-2 S models with different N-glycans at key functional sites, namely N234, N165 and N343. We also assessed the effect of a change in the SARS-CoV-2 S glycan shield’s topology at N370, due to the recently acquired T372A mutation. Our results indicate that the structures of the N-glycans at N234, N165 and N343 affect the stability of the active (or open) S conformation, and thus its exposure and accessibility. Furthermore, while glycosylation at N370 stabilizes the open S conformation, we find that the N370 glycan binds the closed receptor binding domain (RBD) surface, essentially tying the closed protomers together. These results suggest that the loss of the N370 glycosylation site in SARS-CoV-2 may have increased the availability of the open S form, perhaps contributing to its higher infectivity relative to CoV1 and other variants carrying the sequon. Finally, we discuss these specific changes to the topology of the SARS-CoV-2 S glycan shield through ancestral sequence reconstruction of select SARS strains and discuss how they may have evolved to affect S activity.