Beyond Shielding: The Roles of Glycans in the SARS-CoV‑2\nSpike Protein

Abstract

The ongoing COVID-19\npandemic caused by severe acute respiratory\nsyndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 28,000,000\ninfections and 900,000 deaths worldwide to date. Antibody development\nefforts mainly revolve around the extensively glycosylated SARS-CoV-2\nspike (S) protein, which mediates host cell entry by binding to the\nangiotensin-converting enzyme 2 (ACE2). Similar to many other viral\nfusion proteins, the SARS-CoV-2 spike utilizes a glycan shield to\nthwart the host immune response. Here, we built a full-length model\nof the glycosylated SARS-CoV-2 S protein, both in the open and closed\nstates, augmenting the available structural and biological data. Multiple\nmicrosecond-long, all-atom molecular dynamics simulations were used\nto provide an atomistic perspective on the roles of glycans and on\nthe protein structure and dynamics. We reveal an essential structural\nrole of <i>N</i>-glycans at sites N165 and N234 in modulating\nthe conformational dynamics of the spike’s receptor binding\ndomain (RBD), which is responsible for ACE2 recognition. This finding\nis corroborated by biolayer interferometry experiments, which show\nthat deletion of these glycans through N165A and N234A mutations significantly\nreduces binding to ACE2 as a result of the RBD conformational shift\ntoward the “down” state. Additionally, end-to-end accessibility\nanalyses outline a complete overview of the vulnerabilities of the\nglycan shield of the SARS-CoV-2 S protein, which may be exploited\nin the therapeutic efforts targeting this molecular machine. Overall,\nthis work presents hitherto unseen functional and structural insights\ninto the SARS-CoV-2 S protein and its glycan coat, providing a strategy\nto control the conformational plasticity of the RBD that could be\nharnessed for vaccine development.