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

TLDR

The COVID‑19 pandemic, caused by SARS‑CoV‑2, has infected over 28 million people and killed 900 k worldwide, and the heavily glycosylated spike protein—central to host cell entry via ACE2—uses a glycan shield to evade immunity. The study aims to construct a full‑length, glycosylated model of the SARS‑CoV‑2 spike protein in both open and closed conformations to enhance structural and biological understanding. All‑atom, microsecond‑long molecular dynamics simulations and end‑to‑end accessibility analyses were performed to examine glycan roles and spike structure and dynamics. The study shows that N‑glycans at N165 and N234 stabilize the receptor‑binding domain in the up conformation, and their removal shifts the RBD toward the down state, reducing ACE2 binding, thereby revealing glycan‑mediated control of spike plasticity that could inform vaccine design.

Abstract

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.