Predicted binding interface between coronavirus nsp3 and nsp4

  1. Zach Hensel

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Abstract

Double membrane vesicles (DMVs) in coronavirus-infected cells feature pores that span both membranes. DMV pores were observed to have six-fold symmetry and include the nsp3 protein. Co-expression of SARS-CoV nsp3 and nsp4 induces DMV formation, and elements of nsp3 and nsp4 have been identified that are essential for membrane disruption. I describe a predicted luminal binding interface between nsp3 and nsp4 that is membrane-associated, conserved in SARS-CoV-2 during the COVID-19 pandemic and in diverse coronaviruses, and stable in molecular dynamics simulation. Combined with structure predictions for the full-length nsp4 monomer and cryo-EM data, this suggests a DMV pore model in which nsp4 spans both membranes with nsp3 and nsp4 inserted into the same bilayer. This approach may be able to identify additional protein-protein interactions between coronavirus proteins.

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    SciScore for 10.1101/2022.03.05.483145: (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
    It is defined by the FoldDock scripts “fetch_plDDT.py” and “vis_analyze.py” as: Parameters used for calculating pDockQ were retrieved from the FoldDock GitLab repository (9 February 2022), and pDockQ was calculated using a PyMOL script.
    pDockQ
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequences and their UniProtKB identifiers are shown in Table 1.
    UniProtKB
    suggested: (UniProtKB, RRID:SCR_004426)
    Following the FoldDock scripts, interface residues were multiply counted both for calculating IF_plDDT and N if they had multiple interface contacts.
    FoldDock
    suggested: None
    Figures were arranged and rendered using VMD (Humphrey, Dalke, and Schulten 1996) or Pymol (DeLano 2002).
    Pymol
    suggested: (PyMOL, RRID:SCR_000305)
    Distograms were adapted from those output by ColabFold or made using NanoHUB (Madhavan et al. 2013).
    NanoHUB
    suggested: (nanoHUB, RRID:SCR_013963)
    Multiple sequence alignments were prepared from full-length nsp3 and nsp4 sequences using Clustal Omega (Sievers et al. 2011), phylogenetic trees were generated from alignments using Simple Phylogeny (ClustalW2), and tree images were generated using iTOL (Letunic and Bork 2021), all with default settings apart from formatting.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Sequence logos for nsp3 and nsp4 regions considered in molecular dynamics simulations were generated using WebLogo 3 (Crooks et al. 2004) after acquiring multiple sequence alignments with MMseqs2 following the same workflow used for structure predictions (Steinegger and Söding 2017).
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A limitation of this approach is that it does not predict post-translational modifications. For example, it was observed that mutation of glycosylated nsp4 residues induced defects in viral replication (Gadlage et al. 2010), and this may indicate protein-protein interactions or monomer structures that cannot be accurately predicted in the absence of accounting for post-translational modifications. An additional limitation is that molecular dynamics simulations here are still relatively short, and it is likely that these nsp3 and/or nsp4 regions would have additional protein-protein interactions in the context of a DMV pore. While some interactions are predicted for additional transmembrane nsp3 regions, it was not possible to construct a larger complex with any confidence. Perhaps additional sequence sequence information acquired during the pandemic can be used to improve predictions for SARS-CoV-2 structures and protein-protein interactions. Lastly, identification of a short nsp3 peptide that may be essential for DMV pore formation suggests a possible therapeutic approach. However, a drug mimicking this region of nsp3 would have to overcome the apparent strength of the interface (Table 2), ER permeability, and likelihood of evolving resistance.

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

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  2. Version published to 10.1101/2022.03.05.483145v1 on bioRxiv