N501Y mutation of spike protein in SARS-CoV-2 strengthens its binding to receptor ACE2

  1. Fang Tian
  2. Bei Tong
  3. Liang Sun
  4. Shengchao Shi
  5. Bin Zheng
  6. Zibin Wang
  7. Xianchi Dong
  8. Peng Zheng

  • Curated by eLife

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    Evaluation Summary:

    Tian et al. used a battery of biophysical techniques to compare wild-type SARS-CoV-2 with two variants (B.1.1.7 and B.1.351). The authors obtained evidence consistent with the receptor-binding domain (RBD) of the variants exhibiting higher affinity ACE2, providing a possible explanation for their higher transmission. However, the effects are modest, and further work will be needed to confirm that this difference explains the increased transmission of the variants. Despite the need for follow-up studies, the paper is among the first to show definitively that the N501Y mutation confers enhanced binding to ACE2.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

SARS-CoV-2 has been spreading around the world for the past year. Recently, several variants such as B.1.1.7 (alpha), B.1.351 (beta), and P.1 (gamma), which share a key mutation N501Y on the receptor-binding domain (RBD), appear to be more infectious to humans. To understand the underlying mechanism, we used a cell surface-binding assay, a kinetics study, a single-molecule technique, and a computational method to investigate the interaction between these RBD (mutations) and ACE2. Remarkably, RBD with the N501Y mutation exhibited a considerably stronger interaction, with a faster association rate and a slower dissociation rate. Atomic force microscopy (AFM)-based single-molecule force microscopy (SMFS) consistently quantified the interaction strength of RBD with the mutation as having increased binding probability and requiring increased unbinding force. Molecular dynamics simulations of RBD–ACE2 complexes indicated that the N501Y mutation introduced additional π-π and π-cation interactions that could explain the changes observed by force microscopy. Taken together, these results suggest that the reinforced RBD–ACE2 interaction that results from the N501Y mutation in the RBD should play an essential role in the higher rate of transmission of SARS-CoV-2 variants, and that future mutations in the RBD of the virus should be under surveillance.

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  1. Version published to 10.7554/elife.69091 on eLife
  2. eLife

    Evaluation Summary:

    Tian et al. used a battery of biophysical techniques to compare wild-type SARS-CoV-2 with two variants (B.1.1.7 and B.1.351). The authors obtained evidence consistent with the receptor-binding domain (RBD) of the variants exhibiting higher affinity ACE2, providing a possible explanation for their higher transmission. However, the effects are modest, and further work will be needed to confirm that this difference explains the increased transmission of the variants. Despite the need for follow-up studies, the paper is among the first to show definitively that the N501Y mutation confers enhanced binding to ACE2.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Tian et al. used a battery of biophysical techniques to compare wild-type SARS-CoV-2 with three variants (B.1.1.7, B.1.351, and P.1). This work is significant because these variants are more transmittable to humans than wild-type. Strikingly, these mutants all share a common mutation, N501Y. Understanding the mechanism that renders these mutants more transmittable can inform the design and development of effective therapeutic strategies against the variants. A significant strength of this work is that Tian et al. were indeed able to discern a difference in the biophysical properties of mutants. N501Y appears to increase the affinity receptor-binding domain (RBD) and ACE2. They found evidence that this increase may be a result of decreased RBD-ACE2 dissociation rate. However, the effects are modest, and further work will be needed to confirm that this difference the increased transmission of the variants.

  4. eLife

    Reviewer #2 (Public Review):

    They perform cell-based binding experiments, surface plasmon resonance affinity measurements, AFM rupture force measurements and molecular dynamics simulations to characterize the binding strength for N501Y, and the triple mutant (N501Y / K417N / E484K). They conclude from these studies that the N501Y mutation in RBD has the most significant role in enhancing the binding to ACE2 and finally comment on the significance of this finding on the efficacy of currently approved vaccine formulations.

    The goal was to characterize at the molecular level the influence of these observed mutations in RBD on ACE2 receptor binding affinity and rupture forces. The paper's strengths are that the experiments and systems are carefully designed. The main weakness is that the increased affinity of N501Y was already reported, therefore the paper does not present a truly new finding (https://www.nature.com/articles/s41422-021-00496-8; https://doi.org/10.1016/j.cell.2020.08.012). Given the current need for valid and reproducible quality studies on SARS-CoV-2, findings that validate prior studies on new / different measurement platforms should be welcomed.

    As far as I know this is the first report of an AFM study of N501Y or the triple mutant. The results do indeed support the conclusion that the variant N501Y confers enhanced ACE2 binding.

  5. ScreenIT

    SciScore for 10.1101/2021.02.14.431117: (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
    All RDB and human ACE2 proteins were expressed in Expi293 cells with OPM-293 CD05 serum-free medium (OPM Biosciences).
    Expi293
    suggested: RRID:CVCL_D615)
    ACE2 expression cell line was constructed by transient transfection of HEK293 cells and used for flow cytometry and AFM in this study.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Briefly, ACE2-mCherry cells were seeded in a poly-D-lysine precoated confocal dish and stained with AlexaFluor488-labeled-RBD (100 nM) for 30 min at room temperature.
    ACE2-mCherry
    suggested: None
    Software and Algorithms
    SentencesResources
    Optical images were analyzed using ImageJ software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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.

    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.

  6. Version published to 10.1101/2021.02.14.431117v2 on bioRxiv
  7. Version published to 10.1101/2021.02.14.431117v1 on bioRxiv