Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity
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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 ACE2-accessible “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.
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SciScore for 10.1101/2020.05.21.109157: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pseudotyped HIV-1 particles expressing the SARS-CoV-2 Spike glycoprotein and NanoLuc luciferase as a reporter were generated by transfection of HEK293T cells with pNL4-3DEnv-NanoLuc and pSARS-CoV2-Strunc. pNL4-3DEnv-NanoLuc was derived from pNL4-3 (Adachi et al., 1986) by inserting a 940 bp deletion after the vpu stop-codon, resulting in loss of Env-expression. HEK293Tsuggested: NoneSupernatants containing virus were harvested and … SciScore for 10.1101/2020.05.21.109157: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Pseudotyped HIV-1 particles expressing the SARS-CoV-2 Spike glycoprotein and NanoLuc luciferase as a reporter were generated by transfection of HEK293T cells with pNL4-3DEnv-NanoLuc and pSARS-CoV2-Strunc. pNL4-3DEnv-NanoLuc was derived from pNL4-3 (Adachi et al., 1986) by inserting a 940 bp deletion after the vpu stop-codon, resulting in loss of Env-expression. HEK293Tsuggested: NoneSupernatants containing virus were harvested and filtered 48 hr post transfection and used for infection of ACE2-overexpressing 293T cells. 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)For experiments, Vero E6 cells were cultured in Minimal Essential Media (MEM), 10% Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources The half maximal inhibitory concentration (IC50) was determined using 4-parameter nonlinear regression (GraphPad Prism). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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