Enhanced Surface Accessibility of SARS-CoV-2 Omicron Spike Protein Due to an Altered Glycosylation Profile

SARS-CoV-2 spike (S) proteins undergo extensive glycosylation, aiding in proper folding, enhancing stability, and evading host immune surveillance. In this study, we used mass spectrometric analysis to elucidate the N-glycosylation characteristics and disulfide bonding of recombinant spike proteins derived from the SARS-CoV-2 Omicron variant (B.1.1.529) in comparison with the D614G spike variant. Furthermore, we conducted microsecond-long molecular dynamics simulations on spike proteins to resolve how the different N-glycans impact spike conformational sampling in the two variants. Our findings reveal that the Omicron spike protein maintains an overall resemblance to the D614G spike variant in terms of site-specific glycan processing and disulfide bond formation. Nonetheless, alterations in glycans were observed at certain N-glycosylation sites. These changes, in synergy with mutations within the Omicron spike protein, result in increased surface accessibility of the macromolecule, including the ectodomain, receptor-binding domain, and N-terminal domain. Additionally, mutagenesis and pull-down assays reveal the role of glycosylation of a specific sequon (N149); furthermore, the correlation of MD simulation and HDX-MS identified several high-dynamic areas of the spike proteins. These insights contribute to our understanding of the interplay between structure and function, thereby advancing effective vaccination and therapeutic strategies.

LysC-Trypsin * The proteins were sequentially digested by listed combination of two enzymes with the first digestion conducted at 52⁰C for 60 min and the second one at 37⁰C overnight.CHTR and aLP represent chymotrypsin and a-lytic protease, respectively.Table S1.N-Glycosylation analysis data of SARS-CoV-2 Omicron and D614G S proteins obtained from the experiments of various digestion conditions.The top row of each sequon was selected for discussion in the text of this report.

Figure S1 .
Figure S1.Workflow of the structural analysis of the SARS-CoV-2 Omicron and D614G spike proteins.

Figure S5 .
Figure S5.Glycan shielding near the fusion peptide.The D614G (A) and Omicron (B) S proteins are depicted in cyan using a cartoon representation.The superimposed glycans are represented by colorful licorice models.These glycan configurations were captured at intervals of 0.25 μs throughout the net 4.2 μs of simulation trajectories for the D614G and Omicron S proteins.Fusion peptides are highlighted in orange.

Figure S6 .
Figure S6.Glycan shielding of the receptor binding domain (RBD) in the SARS-CoV-2 S protein.The residues from S469 to V483 of chain A in the D614G (A) and Omicron (B) S proteins are shown in pink.All glycan residues within 5 Å of the RBDs are depicted in the figures.These structures are superimposed at intervals of 0.25 μs along the respective simulation trajectories.

Figure S7 .
Figure S7.The accessible surface area (ASA) of the N-terminal domain (A) and receptor binding domain (B), factoring in the presence of glycans.The ASA is measured in Å 2 .

Figure S8 .
Figure S8.Glycan shielding of the N-terminal domain (NTD) in the SARS-CoV-2 S protein.The NTDs of chain A in the D614G (A, B) and Omicron (C, D) S proteins are shown in cyan.All glycan residues within 5 Å of the NTDs are depicted in the figures.These structures are superimposed at intervals of 0.25 μs along the respective simulation trajectories.

Figure S9 .
Figure S9.Full-length glycan coverage at positions N17 and N149.Glycans are depicted in their complete length.The displayed structures are cumulative snapshots taken at 0.25-μs intervals along the respective simulation trajectories.

Figure S10 .
Figure S10.RBD conformation in the D614G S protein.Two-dimensional kernel density estimate (KDE) plot visualizes the spread of RBD conformations within the trajectories of the un-glycosylated (A) and glycosylated (B) systems, with two collective variables (defined in Methods), a distance and a dihedral angle, characterizing the RBD conformations.

Table S2 .
Peak areas of the disulfide bonded (DB) peptides detected in both SARS-CoV-2 spike proteins of D614G and Omicron variants digested by various proteases.The peptides contains cystine residue but do not form a disulfide bond are included.For example, DB3-free C1 represent a peptide with NEM modified free Cys at 391 position 291.c.The sequence and the amino acid position of the peptides are derived from the D614G spike.Letters in parentheses represent substitutions of Omicron spike protein.