Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains

  1. Tongqing Zhou
  2. Yaroslav Tsybovsky
  3. Adam S. Olia
  4. Jason Gorman
  5. Micah A. Rapp
  6. Gabriele Cerutti
  7. Gwo-Yu Chuang
  8. Phinikoula S. Katsamba
  9. Alexandra Nazzari
  10. Jared M. Sampson
  11. Arne Schon
  12. Pengfei D. Wang
  13. Jude Bimela
  14. Wei Shi
  15. I-Ting Teng
  16. Baoshan Zhang
  17. Jeffrey C. Boyington
  18. Mallika Sastry
  19. Tyler Stephens
  20. Jonathan Stuckey
  21. Shuishu Wang
  22. Richard A. Friesner
  23. David D. Ho
  24. John R. Mascola
  25. Lawrence Shapiro
  26. Peter D. Kwong

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Abstract

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. Antibodies can engage RBD but some, such as CR3022, fail to inhibit entry despite nanomolar spike affinity. Here we show the SARS-CoV-2 spike to have low unfolding enthalpy at serological pH and up to 10-times more unfolding enthalpy at endosomal pH, where we observe significantly reduced CR3022 affinity. Cryo-EM structures -at serological and endosomal pH- delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the 'up' conformation. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning and spike shedding of antibodies like CR3022. An endosomal mechanism involving spike-conformational change can thus facilitate immune evasion from RBD-'up'-recognizing antibody.

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  1. Version published to 10.1101/2020.07.04.187989v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.07.04.187989: (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.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    RESOURCE AVAILABILITY: EXPERIMENTAL MODEL AND SUBJECT DETAILS: METHOD DETAILS: QUANTIFICATION AND STATISTICAL ANALYSIS: The BLI and DSC data were analyzed and plotted using Excel and GraphPad Prism.
    Excel
    suggested: None
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The SPR data were processed and fit using Scrubber 2.0 (BioLogic Software).
    Scrubber
    suggested: (Scrubber2, RRID:SCR_015745)
    Cryo-EM data were processed and analyzed using CryoSparc and Relion.
    CryoSparc
    suggested: (cryoSPARC, RRID:SCR_016501)
    Cryo-EM structural statistics were analyzed with Phenix and Molprobity.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

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  3. Version published to 10.1101/2020.07.04.187989v1 on bioRxiv