GotGlycans: Role of N343 Glycosylation on the SARS-CoV-2 S RBD Structure and Co-Receptor Binding Across Variants of Concern (preprint)

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and to structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work we show through over 45 s of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how the structural role of this glycan changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2) and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.

Natural variations within the glycan shield of SARS-CoV-2 impact viral spike dynamics (preprint)

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the glycosylation of a recombinant viral spike mimetic of the P.1 (Gamma) strain, which exhibits two additional N-linked glycan sites compared to the equivalent mimetic of the Wuhan strain. We determine the site-specific glycosylation of these variants and investigate the impact of these glycans by molecular dynamics. The N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the antigenic surface.

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

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, stabilising 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 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.

The oligomannose N-glycans 3D architecture and its response to the FcγRIIIa structural landscape (preprint)

Oligomannoses are evolutionarily the oldest class of N-glycans, where the arms of the common pentasaccharide unit, i.e. Man α1-6)-[Manα(1-3)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ1-Asn, are functionalized exclusively with branched arrangements of mannose (Man) monosaccharide units. In mammalian species oligomannose N-glycans can have up to 9 Man, meanwhile structures can grow to over 200 units in yeast mannan. The highly dynamic nature, branching complexity and 3D structure of oligomannoses have been recently highlighted for their roles in immune escape and infectivity of enveloped viruses, such as HIV-1 and SARS-CoV2. The architectural features that allow these N-glycans to perform their functions is yet unclear, due to their intrinsically disordered nature that hinders their structural characterization. In this work we will discuss the results of over 54 µs of cumulative sampling by molecular dynamics (MD) simulations of differently processed, free (not protein-linked) oligomannose N-glycans common in vertebrates. We then discuss the effects of a protein surface on their structural equilibria based on over 4 µs cumulative MD sampling of the fully glycosylated CD16a Fc gamma receptor (FcγRIIIa), where the type of glycosylation is known to modulate its binding affinity for IgG1s, regulating the antibody-dependent cellular cytotoxicity (ADCC). Our results show that the protein’s structural constraints shift the oligomannoses conformational ensemble to promote conformers that satisfy the steric requirements and hydrogen bonding networks demanded by the protein’s surface landscape. More importantly, we find that the protein does not actively distort the N-glycans into structures not populated in the unlinked forms in solution. Ultimately, the highly populated conformations of the Man5 linked glycans support experimental evidence of high levels of hybrid complex forms at N45 and show a specific presentation of the arms at N162, which may be involved in mediating binding affinity to the IgG1 Fc.

Citizen Scientists Create an Exascale Computer to Combat COVID-19 (preprint)

The SARS-CoV-2/COVID-19 pandemic continues to threaten global health and socioeconomic stability. Experiments have revealed snapshots of many of the viral components but remain blind to moving parts of these molecular machines. To capture these essential processes, over a million citizen scientists have banded together through the Folding@home distributed computing project to create the world’s first Exascale computer and simulate protein dynamics. An unprecedented 0.1 seconds of simulation of the viral proteome reveal how the spike complex uses conformational masking to evade an immune response, conformational changes implicated in the function of other viral proteins, and ‘cryptic’ pockets that are absent in experimental snapshots. These structures and mechanistic insights present new targets for the design of therapeutics.This living document will be updated as we perform further analysis and make the data publicly accessible.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Shielding and Beyond: The Roles of Glycans in SARS-CoV-2 Spike Protein (preprint)

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 7,000,000 infections and 400,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). In the context of vaccine design, similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the glycan shield and the protein structure, stability, and dynamics. End-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which can be harnessed for vaccine development. In addition, a dynamic analysis of the main antibody epitopes is provided. Finally, beyond shielding, a possible structural role of N-glycans at N165 and N234 is hypothesized to modulate and stabilize the conformational dynamics of the spikes receptor binding domain, which is responsible for ACE2 recognition. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, which may be exploited by therapeutic efforts targeting this essential molecular machine.