Towards Targeting the Disordered SARS-CoV-2 Nsp2 C-terminal Region: Partial Structure and Dampened Mobility Revealed by NMR Spectroscopy

  1. Miguel Mompeán
  2. Miguel Á. Treviño
  3. Douglas V. Laurents

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Abstract

Intrinsically disordered proteins (IDPs) play essential roles in regulating physiological processes in eukaryotic cells. Many virus use their own IDPs to “hack” these processes to disactive host defenses and promote viral growth. Thus, viral IDPs are attractive drug targets. While IDPs are hard to study by X-ray crystallography or cryo-EM, atomic level information on their conformational perferences and dynamics can be obtained using NMR spectroscopy. SARS-CoV-2 Nsp2 interacts with human proteins that regulate translation initiation and endosome vesicle sorting, and the C-terminal region of this protein is predicted to be disordered. Molecules that block these interactions could be valuable leads for drug development. To enable inhibitor screening and to uncover conformational preferences and dynamics, we have expressed and purified the 13 C, 15 N-labeled C-terminal region of Nsp2. The 13 Cβ and backbone 13 CO, 1 HN, 13 Cα and 15 N nuclei were assigned by analysis of a series of 2D 1 H- 15 N HSQC and 13 C- 15 N CON as well as 3D HNCO, HNCA, CBCAcoNH and HncocaNH spectra. Overall, the chemical shift data confirm that this region is chiefly disordered, but contains two five-residue segments that adopt a small population of β-strand structure. Whereas the region is flexible on ms/ms timescales as gauged by T 1ρ measurements, the { 1 H}- 15 N NOEs reveal a flexibility on ns/ps timescales that is midway between a fully flexible and a completely rigid chain.

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    SciScore for 10.1101/2020.11.09.374173: (What is this?)

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

    Table 1: Rigor

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    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Peak integrals were measured using Topspin 4.0.8.
    Topspin
    suggested: (TopSpin, RRID:SCR_014227)
    R1ρ relaxation rates were obtained by using the program Kaleidagraph (Synergy Software, version 3.6) to fit an exponential equation: I(t) = Io· exp(−k·t) + I∞, where I(t) is the peak integral at time t, k is the rate, and I∞ is the intensity at infinite time.
    Kaleidagraph
    suggested: (KaleidaGraph, RRID:SCR_014980)

    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 10. 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.

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  2. Version published to 10.1101/2020.11.09.374173v1 on bioRxiv