Ca 2+ -dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide

  1. George Khelashvili
  2. Ambrose Plante
  3. Milka Doktorova
  4. Harel Weinstein

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Abstract

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the “fusion peptide” (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca 2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca 2+ binding to the SARS-CoV-2-FP, the role of Ca 2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca 2+ -bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca 2+ -bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca 2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca 2+ ions. These findings provide novel mechanistic insights regarding the role of Ca 2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.

STATEMENT OF SIGNIFICANCE

SARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed “fusion peptide” (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca 2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca 2+ -dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

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    SciScore for 10.1101/2020.12.03.410472: (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

    Software and Algorithms
    SentencesResources
    The system was equilibrated with NAMD version 2.12 37 following a multi-step protocol during which the backbone atoms of the SARS-CoV-2-FP as well as Ca2+ ions in the solution were first harmonically constrained and subsequently gradually released in four steps (totaling ~3ns), changing the restrain force constants kF from 1, to 0.5, to 0.1 kcal/ (mol Å2), and 0 kcal/ (mol Å2).
    NAMD
    suggested: (NAMD, RRID:SCR_014894)
    After this phase, the velocities of all atoms of the system were reset, and ensemble MD runs were initiated with OpenMM version 7.4.
    OpenMM
    suggested: (OpenMM, RRID:SCR_000436)

    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: We did not find any issues relating to colormaps.

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    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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    • No protocol registration statement was detected.

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  2. Version published to 10.1101/2020.12.03.410472 on bioRxiv