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

Abstract

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 angiotensinconverting 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 spike's 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. Glycan Shield of the Receptor Binding DomainAs discussed in the previous section, the glycan shield plays a critical role in hiding the S protein surface from molecular recognition. However, to effectively function, the spike needs to recognize and bind to ACE2 receptors as the primary host cell infection route. For this reason, the RBM must become fully exposed and accessible. 48 In this scenario, the glycan shield works in concert with a large conformational change that allows the RBD to emerge above the N-glycan coverage. Here, we quantify the ASA of the RBM within RBD-A, corresponding to the RBD/ACE2-interacting region (residues 400-508), at various probe radii in both the Open and Closed systems (Figures 3A and 3D, full data in Tables S4-S6.). As expected, the ASA plots show a significant difference between the "down" (Closed) and "up" (Open) RBD conformations, with the RBM area covered by glycans being remarkably larger in the former. When RBD-A is in the "up" conformation, its RBM shows an average (across all radii) of only ~9% surface area covered by glycans, compared with ~35% in the Closed system (Figures 3A and 3D). This difference is further amplified when considering a larger probe radius of 15 Å, with a maximum of 11% and 46% for Open and Closed, respectively. Interestingly, for smaller probes (1.4-3 Å) the shielding becomes weak in both systems, with an average of 6% and 16% for Open and Closed, respectively.Note that the RBD regio...

DC/L-SIGN recognition of spike glycoprotein promotes SARS-CoV-2 trans-infection and can be inhibited by a glycomimetic antagonist

“…DC-SIGN and L-SIGN have been already described as receptor of SARS-CoV-1 and twenty out of the twenty-two SARS-CoV-2 S protein N-linked glycosylation sequons are conserved. Glycan shielding represent 60 to 90 % of the spike surface considering the head or the stalk of the S ectodomain, respectively (Casalino et al, 2020;Sikora et al, 2020). One third of N-glycans of SARS-CoV-2 spike are of the oligomannose type (Watanabe et al, 2020a).…”

“…Recent thorough glycan and structural analyses comparing both SARS-CoV-1/2 spike glycoproteins have shown that glycosylation is mostly conserved in the two proteins, both in position and nature of the glycan exposed (Watanabe et al, 2020a(Watanabe et al, , 2020bWrapp et al, 2020), creating a glycan shield which complicates neutralization by antibodies. Secondly, elegant molecular dynamic simulations suggested how some of the spike glycans may directly modulate the dynamics of the interaction with ACE2, stabilizing the up conformation of the RBD domain (Casalino et al, 2020;Zhao et al, 2020). Finally, and yet unexplored, spike glycans may contribute to infectivity by acting as anchor points for DC-SIGN and L-SIGN on host cells surfaces.…”

A 3.4-Å cryo-EM structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles

“…Since glycosylation of S plays an important role in the viral life cycle and immune-evasion (Casalino et al 2020;Vigerust & Shepherd 2007;Watanabe et al 2019Watanabe et al , 2020, we further investigated the glycosylation sites in our map by computing a difference map between our map and the map of biochemically purified S (EMD-8331). All glycosylation sites previously identified by a combination of cryo-EM densities protruding from the expected amino acid side chains and mass spectroscopy ( Table 1 and Table S2) (Walls et al 2016) were found in our map and annotated in yellow (Fig.…”

The SARS-CoV-2 Spike harbours a lipid binding pocket which modulates stability of the prefusion trimer

“…All three lie close to RBDs in the 'locked' conformation, well positioned to shield the ACE2 binding site from immune recognition. In addition, the first or second glycosidic rings of the N165 and N234 glycans make polar interactions underneath the tip of the RBD, opposite to the ACE2 binding site: GlcNacN165 with Y351' and GlcNacN234 with R457' and S459' (Casalino et al, 2020) ( Figure 1D). We would expect these to be recapitulated in the highly processed sugars of wild-type virus (Watanabe et al, 2020).…”