Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

  1. Valeria Marquez-Miranda
  2. Maximiliano Rojas
  3. Yorley Duarte
  4. Ignacio Diaz-Franulic
  5. Miguel Holmgren
  6. Raul E. Cachau
  7. Fernando D. Gonzalez-Nilo

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Abstract

SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimer’s cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domain’s potential permeation pathway has six constrictions narrowing to about 1 Å in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavity’s electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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    Table 1: Rigor

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    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The system was equilibrated using the six steps scripts provided by the CHARMM-GUI [17–19] webserver under the AMBER molecular package [20–22].
    AMBER
    suggested: (AMBER, RRID:SCR_016151)
    We used Python 3 [33] and the packages NumPy [34], pandas [35], and matplotlib [36] for data analysis.
    Python
    suggested: (IPython, RRID:SCR_001658)
    NumPy
    suggested: (NumPy, RRID:SCR_008633)
    matplotlib
    suggested: (MatPlotLib, RRID:SCR_008624)

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    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.

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    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.

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  2. Version published to 10.1101/2020.10.22.349522v1 on bioRxiv