An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking

  1. Debajit Dey
  2. Suruchi Singh
  3. Saif Khan
  4. Matthew Martin
  5. Nicholas J. Schnicker
  6. Lokesh Gakhar
  7. Brian G. Pierce
  8. S. Saif Hasan

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Abstract

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

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  1. Version published to 10.1038/s42003-022-03063-y
  2. Version published to 10.1101/2021.09.03.458953v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.09.03.458953: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    Protein and peptide production: The S. pombe αCOPI-WD40 domain was synthesized by TOPGENE and cloned in pcDNA3.1(+) with a C-terminal strep-tag for affinity purification.
    pcDNA3.1(+)
    suggested: RRID:Addgene_129020)
    β’COPI-WD40 (residues 1-304) from Saccharomyces cerevisiae was cloned in pGEX-6P-1 vector and expressed overnight in E. coli pLysS cells at 18°C.
    pGEX-6P-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Expression was performed in Expi293 mammalian cells using the Thermo Fisher ExpiFectamine expression kit.
    Thermo Fisher ExpiFectamine
    suggested: None
    Molecular replacement was performed in Phenix using a previously determined αCOPI-WD40 domain structure (PDB ID 4J87) as the search model (McCoy et al., 2007; Rossmann and Blow, 1962).
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Iterative model building and refinement were performed in Phenix.refine (Afonine et al., 2012) and Coot (Emsley and Cowtan, 2004).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Figures were generated in PyMol.
    PyMol
    suggested: (PyMOL, RRID:SCR_000305)
    Analysis of Ramachandran angles: The crystal structures of wild type, Arg57→Ala, and Tyr139→Ala αCOPI-WD40 were analyzed in Molprobity (Williams et al., 2018).
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Default parameters were used, with the exception of extra rotamers allowed during packing of modeled side chains, specified by command line parameters: Sequence analysis of dibasic motifs in the human membrane proteome: UNIPROT identifiers of secreted and membrane-bound human proteins, as well as secreted/membrane-bound protein isoforms, were downloaded from the Human Protein Atlas (http://www.proteinatlas.org) (Thul et al., 2017).
    http://www.proteinatlas.org
    suggested: (HPA, RRID:SCR_006710)

    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.

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  4. Version published to 10.1101/2021.09.03.458953v1 on bioRxiv