Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

Abstract

A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction.Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology. Introduction:In late 2019, a novel, pathogenic coronavirus (SARS-CoV-2) entered the human population and has since spread throughout the world. The number of people suffering from the associated disease (COVID-19) continues to rise, increasing the need for effective therapeutic interventions. SARS-CoV-1 and SARS-CoV-2 Spike proteins are highly homologous (~76% sequence identity). Similar to SARS-CoV-1, the interaction between the SARS-CoV-2 Spike protein and the angiotensinconverting enzyme 2 (ACE2) on human cells is critical for viral entry into host cells (Gralinski & Menachery, 2020;Tai et al., 2020;Wu et al., 2020). SARS-CoV-2 Spike is an obligate trimer, while ACE2 presents as a dimer on the cell surface (Chen, Liu, & Guo, 2020). Several highresolution structures of SARS-CoV-2 Spike receptor binding domain (Spike-RBD) bound to ACE2 have been published (Lan et al., 2020;Yan et al., 2020). However, as of this writing, structures of SARS-CoV-2 Spike trimer in complex with either the dimeric or monomeric form of ACE2 have not been reported, resulting in an incomplete understanding of the nature of this interaction.Structural studies of trimeric SARS-CoV-2 and SARS-CoV-1 Spike protein demonstrate that each of the Spike-RBDs, as in other coronaviruses, can undergo hinge-like movements to transition between "up" or "down" conformations. The...

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

“…Results in Figure S13 were generated from live SARS-CoV-2 neutralization assays performed as previously described (15) at the University of California, San Francisco. Of note, the clinical strain used in the assay was SARS-CoV-2 virus clinical isolate 2019-nCoV/USA-WA1/2020 (BEI resources) and the infection duration was 16 hours instead of 24 hours.…”

“…APN01 is currently in phase II clinical trials in Europe for treatment of SARS-CoV-2 (14) (NCT04335136). However, we and others have shown that WT ACE2 binds the SARS-CoV-2 spike RBD with only modest affinity (KD ~15 nM) (15)(16)(17). ACE2 is therefore a good candidate for affinity optimization, especially because potent blocking antibodies to the spike protein can be isolated with binding affinity (KD) values in the mid-to low-pM range (3,4,6,7,9,(18)(19)(20).…”

ACE2, the Receptor that Enables Infection by SARS‐CoV‐2: Biochemistry, Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators

“…Interestingly, recent work shows that a monomeric form of the PD (ACE2 18-640) binds efficiently to the isolated RBD from spike, but does not significantly bind full-length spike trimers, while the PD (18-640)-Fc dimer can bind full-length spike trimers with reduced on-rate but also reduced off-rate. [60] CryoEM studies with SARS-CoV virions show that it binds to three soluble ACE2-Fc molecules. [61] After interaction with ACE2, the spike trimer undergoes conformational changes that promote membrane fusion.…”

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

“…We first expressed the Spike-RBD (residues 328-533) and the ACE2 peptidase domain (residues 1-640) as biotinylated Fc-fusions for VH-phage selections. 34 To specifically enrich for VH-phage that bind the ACE2 binding site on Spike-RBD, the library was first cleared with the Spike-RBD-Fc/ACE2-Fc complex to remove phage that bind outside the ACE2 binding interface. This was followed by selection on Spike-RBD-Fc alone to enrich for phage that bind the unmasked ACE2 binding site ( Fig.…”

“…Live virus neutralization assays were done as previously described. 34 Relative copy number (RCN) of viral transcript level compared to host transcript was determine using the ∆∆CT method.…”