In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding

  1. Neil Bate
  2. Christos G. Savva
  3. Peter C. E. Moody
  4. Edward A. Brown
  5. Sian E. Evans
  6. Jonathan K. Ball
  7. John W. R. Schwabe
  8. Julian E. Sale
  9. Nicholas P. J. Brindle

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Abstract

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

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  1. Version published to 10.1371/journal.ppat.1010733
  2. ScreenIT

    SciScore for 10.1101/2021.12.23.473975: (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.
    RandomizationIn addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were washed and incubated with anti-FLAG-APC and anti-Histidine6-PE antibodies on ice for 20 mins, followed by washing.
    anti-FLAG-APC
    suggested: None
    anti-Histidine6-PE
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Expression and purification of soluble proteins: HEK 293 cells were transfected with mammalian expression vectors encoding the relevant protein using polyethylenimine and cells incubated for approximately 3 days to allow accumulation of the secreted protein in the medium.
    HEK 293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Flow cytometry: Vero-E6 cells were cultured in DMEM with 10% (v/v) FBS.
    Vero-E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.
    Wuhan-Hu-1 CoV-2 RBD
    suggested: None
    Recombinant DNA
    SentencesResources
    cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.
    pHypermut2
    suggested: None
    In addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Software and Algorithms
    SentencesResources
    Briefly, movies were corrected for motion using MotionCor 2.1.425 and the contrast transfer function parameters were estimated using GCTF 1.1826.
    GCTF
    suggested: (GCTF, RRID:SCR_016500)
    Initial rigid-body docking of the crystal structure (PDB ID 6M0J) was performed using UCSF Chimera 1.1528 and further model building was performed in Coot 0.9.629.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    After manual rebuilding real-space refinement of the coordinates was performed using Phenix 1.19.230.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 26. 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.

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

    About SciScore

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  3. Version published to 10.1101/2021.12.23.473975v1 on bioRxiv