The SARS-CoV-2 spike protein is heavily glycosylated. These post-translational modifications aid in interactions with human angiotensin-converting enzyme 2 (hACE2), assist in antibody evasion, and help facilitate protein dynamics. However, the exact role glycans play in maintaining the protein structure and conformation is not fully understood. In this study, we elucidated the structural function of these polysaccharides in the recombinant 614G HexaPro spike ectodomain protein by PNGase F glycosidase treatment and evaluated the subsequent effects on protein conformational dynamics via hydrogen/deuterium exchange-mass spectrometry. Our results show that enzymatic deglycosylation led to widespread conformational changes, both in regions with glycosylation sites and those without. Notably, the 630 loop, which mediates the receptor-binding domain (RBD) up/down position, became disordered, overcoming the rigidification that resulted from the D614G mutation. Additionally, heptad repeat 2, which plays a role in viral fusion machinery, also exhibited an increase in deuteration. Regions that showed a decrease in deuteration after treatment include subdomain 1, the fusion peptide proximal region, and the coiled coil. We also provide insights into the reduced thermal stability and altered binding behavior of antibodies and hACE2 in addition to changes in deuteration following enzymatic glycan removal.
Osman et al. (Wed,) studied this question.