Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike Dynamics.

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 site-specific glycosylation of recombinant viral spike mimetics of B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.1.529 (Omicron). The P.1 strain exhibits two additional N-linked glycan sites compared to the other variants analyzed and we investigate the impact of these glycans by molecular dynamics. The acquired 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 protein surface.

Fine-tuning the spike: role of the nature and topology of the glycan shield in the structure and dynamics of the SARS-CoV-2 S.

The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer; a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shield's topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that N-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, and potentially increases host cell surface localization.

SARS-CoV-2 simulations go exascale to predict dramatic spike opening and cryptic pockets across the proteome.

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression and replication that depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate 0.1 seconds of the viral proteome. Our adaptive sampling simulations predict dramatic opening of the apo spike complex, far beyond that seen experimentally, explaining and predicting the existence of 'cryptic' epitopes. Different spike variants modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also discover dramatic conformational changes across the proteome, which reveal over 50 'cryptic' pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

Oligomannose N-Glycans 3D Architecture and Its Response to the FcγRIIIa Structural Landscape.

Oligomannoses are evolutionarily the oldest class of N-glycans, where the arms of the common pentasaccharide unit, i.e., Manα(1-6)-[Manα(1-3)]-Manß(1-4)-GlcNAcß(1-4)-GlcNAcß1-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 are 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 γ 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.

Beyond Shielding: The Roles of Glycans in the SARS-CoV-2 Spike Protein.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000 infections and 900,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viral fusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of the 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 roles of glycans and on the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift toward the "down" state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of the SARS-CoV-2 S protein, which may be exploited in the therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.

How and why plants and human N-glycans are different: Insight from molecular dynamics into the "glycoblocks" architecture of complex carbohydrates.

The N-glycosylation is one of the most abundant and diverse post-translational modifications of proteins, implicated in protein folding and structural stability, and mediating interactions with receptors and with the environment. All N-glycans share a common core from which linear or branched arms stem from, with functionalization specific to different species and to the cells' health and disease state. This diversity generates a rich collection of structures, all diversely able to trigger molecular cascades and to activate pathways, which also include adverse immunogenic responses. These events are inherently linked to the N-glycans' 3D architecture and dynamics, which remain for the large part unresolved and undetected because of their intrinsic structural disorder. In this work we use molecular dynamics (MD) simulations to provide insight into N-glycans' 3D structure by analysing the effects of a set of very specific modifications found in plants and invertebrate N-glycans, which are immunogenic in humans. We also compare these structural motifs and combine them with mammalian N-glycan motifs to devise strategies for the control of the N-glycan 3D structure through sequence. Our results suggest that the N-glycans' architecture can be described in terms of the local spatial environment of groups of monosaccharides. We define these "glycoblocks" as self-contained 3D units, uniquely identified by the nature of the residues they comprise, their linkages and structural/dynamic features. This alternative description of glycans' 3D architecture can potentially lead to an easier prediction of sequence-to-structure relationships in complex carbohydrates, with important implications in glycoengineering design.

SARS-CoV-2 Simulations Go Exascale to Capture Spike Opening and Reveal Cryptic Pockets Across the Proteome.

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of 'cryptic' epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 'cryptic' pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

Beyond Shielding: The Roles of Glycans in SARS-CoV-2 Spike Protein.

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 15,000,000 infections and 600,000 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). 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 roles of glycans, and the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spike's receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift towards the "down" state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which may be exploited by therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.