Interaction of Human ACE2 to Membrane-Bound SARS-CoV-1 and SARS-CoV-2 S Glycoproteins

Abstract

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is responsible for the current global coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The viral entry of SARS-CoV-2 depends on an interaction between the receptor-binding domain of its trimeric spike glycoprotein and the human angiotensin-converting enzyme 2 (ACE2) receptor. A better understanding of the spike/ACE2 interaction is still required to design anti-SARS-CoV-2 therapeutics. Here, we investigated the degree of cooperativity of ACE2 within both the SARS-CoV-2 and the closely related SARS-CoV-1 membrane-bound S glycoproteins. We show that there exist differential inter-protomer conformational transitions between both spike trimers. Interestingly, the SARS-CoV-2 spike exhibits a positive cooperativity for monomeric soluble ACE2 binding when compared to the SARS-CoV-1 spike, which might have more structural restraints. Our findings can be of importance in the development of therapeutics that block the spike/ACE2 interaction.

Keywords: ACE2-Fc; COVID-19; CR3022 antibody; Coronavirus; SARS-CoV-1; SARS-CoV-2; human ACE2 receptor; neutralization; spike glycoproteins.

Figure 1

Differences between severe acute respiratory syndrome virus 1 (SARS-CoV-1) and severe acute respiratory syndrome virus 2 (SARS-CoV-2) spikes in their abilities to engage soluble monomeric angiotensin-converting enzyme 2 (sACE2), ACE2-Fc, and CR3022. The binding of (A) sACE2, (B) ACE2-Fc, and (C) CR3022 to SARS-CoV-1 or SARS-CoV-2 (wt or D614G) spikes expressed on the cell surface were measured by flow cytometry. Increasing concentrations of each ligand were incubated with Spike-expressing cells as described in the Material and Methods. Means ± SEM derived from at least three independent experiments are shown. The Hill coefficient was determined using GraphPad software.

Figure 2

Sensitivity of viruses harboring SARS-CoV-1 S and SARS-CoV-2 spikes to neutralization by sACE2, ACE2-Fc, and CR3022. Pseudoviral particles coding for the luciferase reporter gene and bearing the following glycoproteins: SARS-CoV-2 (wt or D614G) S, SARS-CoV-1 S, or VSV-G were used to infect 293T-ACE2 cells. Pseudoviruses were incubated with increasing concentrations of (A) sACE2, (B) ACE2-Fc, and (C) CR3022 at 37 °C for 1 h prior to infection of 293T-ACE2 cells. Means ± SEM derived from at least two independent experiments are shown. (D) Neutralization by sACE2 was correlated with the sACE2 binding quantified by flow cytometry to SARS-CoV-1 and SARS-CoV-2 spikes. (E) Model indicating the stoichiometry needed for neutralization by sACE2 to either SARS-CoV-1 S-or SARS-CoV-2 (wt or D614G) S-bearing pseudovirions.

Figure 3

Proposed energy landscapes of spike trimer opening of SARS-CoV-2 and SARS-CoV-1. We assume conformational landscapes for both species of spikes (SARS-CoV-1: black; SARS-CoV-2: red) that permit equilibration among four distinct states: all- receptor-binding-domains (RBDs) -down (“0”), one-RBD-up (“1”), two-RBDs-up (“2”), and three-RBDs-up (“3”). We hypothesize that the SARS-CoV-2 spike energetic barrier for transitioning from all-RBDs-down to one-RBD-up ($E_{01}^{*}$) is lower for SARS-CoV-2 than for SARS-CoV-1, while both experience the same barrier for the reverse transition, making the one-RBD-up state more stable for SARS-CoV-2 than for SARS-CoV-1, as illustrated by the relative energy differences $\Delta E_{01}$. However, SARS-CoV-1 spike likely needs to overcome substantially higher energy barriers to transit from the one-RBD-up state to the two-RBDs-up state ($E_{12}^{*}$), as compared with SARS-CoV-2 spike. This underlying conformational selection mechanism results in a larger population of SARS-CoV-2 spike in two or three RBDs up state and has higher chance to engage multiple ACE2, which eventually spurs the complete open and dissociation of S1 from S2 and induces membrane fusion. Cartoon representation of closed, one-RBD-up, two-RBDs-up states of spike were generated from deposited structures in Protein Data Bank (6ZWV, 6VSB, 6 × 2B, respectively). The three-RBDs-up model was generated by C3 symmetry superposition of three one-RBD-up protomer in 6VSB.