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

  1. Irene Lui
  2. Xin X. Zhou
  3. Shion A. Lim
  4. Susanna K. Elledge
  5. Paige Solomon
  6. Nicholas J. Rettko
  7. Beth Shoshana Zha
  8. Lisa L. Kirkemo
  9. Josef A. Gramespacher
  10. Jia Liu
  11. Frauke Muecksch
  12. Julio Cesar Cetrulo Lorenzi
  13. Fabian Schmidt
  14. Yiska Weisblum
  15. Davide F. Robbiani
  16. Michel C. Nussenzweig
  17. Theodora Hatziioannou
  18. Paul D. Bieniasz
  19. Oren S. Rosenburg
  20. Kevin K. Leung
  21. James A. Wells

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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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  1. ScreenIT

    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 Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    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.
    HEK293T
    suggested: None
    Supernatants containing virus were harvested and filtered 48 hr post transfection and used for infection of ACE2-overexpressing 293T cells.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    For experiments, Vero E6 cells were cultured in Minimal Essential Media (MEM), 10%
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    The half maximal inhibitory concentration (IC50) was determined using 4-parameter nonlinear regression (GraphPad Prism).
    GraphPad
    suggested: (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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.05.21.109157v1 on bioRxiv