Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike

Abstract

Binding of the spike protein of SARS-CoV-2 to the human angiotensin-converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds to a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.

Keywords: ACE2 receptor; SARS-CoV-2; glycosylation; molecular dynamics; virus-host interaction.

Fig. 1.

MD simulations of fully glycosylated B⁰AT1–ACE2–RBD complex. (A) System setup of the complex with explicit water and physiological concentration of ions. B⁰AT1, ACE2, and the RBD are shown in blue, green, and magenta cartoon, and glycans as licorice. (B) Distinct glycosylation patterns used in the simulations: two variants of homogeneous N-glycosylation (Left) and three variants of heterogeneous glycosylation (Right).

Fig. 2.

Interaction of ACE2 glycans with SARS-CoV-2 spike RBD. (A) Interaction energy between the ACE2 glycans and the RBD. The box for each glycan and MD setup shows the lower to upper quartile values of the data. The whiskers show the range of the data. The orange line is the median of the data. (B) Average number of residue–residue contacts between the N322 glycans and the RBD residues. Color shading highlights the four main interaction regions. (C) Simulation ensemble of the N322 glycan interacting with the RBD (from the HMC_WRBD_WBAT simulation). (D) Close-up of the interaction between the N322 glycan and the RBD in a representative snapshot of C.

Fig. 3.

Interaction of ACE2 glycans with RBD binding site. (A) Interaction energy between the ACE2 glycans and the RBD binding site in the absence of the RBD. The box for each glycan and MD setup shows the lower to upper quartile values of the data. The whiskers show the range of the data. The orange line is the median of the data. (B) Fraction of RBD binding-site area covered by the N90 glycan from SASA calculations using a 5-Å probe for the HMC_WoRBD_WBAT setup. Results for 1.4- and 10-Å probe sizes are shown in SI Appendix, Fig. S11. (C) RBD binding site shielded by the N90 glycan. The RBD binding site, the area shielded by the N90 glycan, and their overlap are colored purple, cyan, and red, respectively. (D) Ensemble of the N90 glycan during the 1-µs-long simulation interacting with the RBD binding site in the HMC_WoRBD_WBAT setup. Steric clashes between glycan and RBD are illustrated by superimposing the RBD according to the HMC_WRBD_WBAT simulation. The RBD binding site is colored yellow. The glycans are shown in sticks. Glycans clashing with the RBD are colored red, and those without clashes are colored cyan.

Fig. 4.

N322 binding site in RBD. (A) Sequence alignment of the N322 binding site in different coronaviruses. The red arrow indicates the difference between SARS-CoV and SARS-CoV-2 in the glycosylation motif of the N370^CoV2 site. (B) Superimposition of antibody CR3022 (brown) targeting the cryptic epitope with a representative snapshot of the N322 glycan (space filling, green). (C) Superimposition of antibody VHH-72 (light blue) targeting the region near the cryptic epitope with a representative snapshot of the N322 glycan (space filling, green). (D) Ensemble of an artificially added N370^CoV2 glycan (sticks) interacting with the N322 binding site (green) in the simulation of the RBD alone.