A Tethered Ligand Assay to Probe SARS-CoV-2:ACE2 Interactions

  1. Magnus S. Bauer
  2. Sophia Gruber
  3. Adina Hausch
  4. Lukas F. Milles
  5. Thomas Nicolaus
  6. Leonard C. Schendel
  7. Pilar López Navajas
  8. Erik Procko
  9. Daniel Lietha
  10. Rafael C. Bernardi
  11. Hermann E. Gaub
  12. Jan Lipfert

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Abstract

SARS-CoV-2 infections are initiated by attachment of the receptor-binding domain (RBD) on the viral Spike protein to angiotensin-converting enzyme-2 (ACE2) on human host cells. This critical first step occurs in dynamic environments, where external forces act on the binding partners and multivalent interactions play critical roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using magnetic tweezers and atomic force spectroscopy as highly complementary single-molecule force spectroscopy techniques, we investigate the RBD:ACE2 interaction over the whole physiologically relevant force range. We combine the experimental results with steered molecular dynamics simulations and observe and assign fully consistent unbinding and unfolding events across the three techniques, enabling us to establish ACE2 unfolding as a molecular fingerprint. Measuring at forces of 2-5 pN, we quantify the force dependence and kinetics of the RBD:ACE2 bond in equilibrium. We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical stability, larger binding free energy, and a lower dissociation rate in comparison to SARS-CoV-1, which helps to rationalize the different infection patterns of the two viruses. By studying how free ACE2 outcompetes tethered ACE2, we show that our assay is sensitive to prevention of bond formation by external binders. We expect our results to provide a novel way to investigate the roles of mutations and blocking agents for targeted pharmaceutical intervention.

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  1. ScreenIT

    SciScore for 10.1101/2021.08.08.455468: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To test blocking, recombinant human ACE2 (Gln18-Ser740, C-terminal His-tag) from RayBiotech (∼3.8 µM in measurement buffer, # 230-30165-100 distributed by antibodies-online GmbH, # ABIN6952473) was flushed into the flowcell and shortly incubated before the measurement.
    ACE2
    suggested: None
    His-tag
    suggested: None
    antibodies-online GmbH,
    suggested: None
    Software and Algorithms
    SentencesResources
    , Experimental Procedures, and Data Analysis: SARS-CoV-1 and SARS-CoV-2
    SARS-CoV-2
    suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
    Equilibrium measurements with MT and rupture experiments with an AFM were evaluated with custom MATLAB and Python scripts to deduce force stability and kinetics.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    Python
    suggested: (IPython, RRID:SCR_001658)

    Results from OddPub: Thank you for sharing your data.

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2021.08.08.455468v1 on bioRxiv