Human 14-3-3 Proteins Site-selectively Bind the Mutational Hotspot Region of SARS-CoV-2 Nucleoprotein Modulating its Phosphoregulation

  1. Kristina V. Tugaeva
  2. Andrey A. Sysoev
  3. Anna A. Kapitonova
  4. Jake L.R. Smith
  5. Phillip Zhu
  6. Richard B. Cooley
  7. Alfred A. Antson
  8. Nikolai N. Sluchanko

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Abstract

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  1. Version published to 10.1016/j.jmb.2022.167891
  2. Version published to 10.1101/2021.12.23.474009v3 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.12.23.474009: (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
    The resulting PCR product containing the S197TAG substitution and carrying the C-terminal cleavable His6-tag was treated with NdeI/HindIII restriction endonucleases and then cloned into a pRBC_14-3-3γ/His6SUMO_mBAD43-204 vector 30 pretreated with the same endonucleases.
    pRBC_14-3-3γ/His6SUMO_mBAD43-204
    suggested: None
    The C-terminal His6-tag enabled the use of the phosphoserine-incorporating E.coli system [BL21 ΔserB(DE3) cells transformed by the pKW2-EFSep plasmid (chloramphenicol resistance) and the target N plasmid] having the Release Factor 1 (responsible for translation termination at TAG codons) 51.
    pKW2-EFSep
    suggested: None
    The SARS-CoV N-containing pGEX plasmid (ampicillin resistance) was kindly provided by Prof. Fulvio Reggiori (University of Groningen, The Netherlands)
    pGEX
    suggested: RRID:Addgene_18100)
    Untagged full-length human 14-3-3γ (Uniprot ID P61981) cloned into a pET21 vector (ampicillin resistance) 52, the surface entropy reduction mutants 53 of the C-terminally truncated human 14-3-3σ (residues 1-231), i.e. Clu1 (159KKE161 → 159AAA161) and Clu3 (75EEK77 → 75AAA77), each containing an N-terminal 3C protease cleavable His6-tag and cloned into the pET28 vector (kanamycin resistance) have been described previously 37.
    pET21
    suggested: RRID:Addgene_90313)
    pET28
    suggested: RRID:Addgene_21766)
    To ensure phosphorylation of SARS-CoV-2 N, its S197L mutant, or SARS-CoV N in E.coli cells, target proteins were co-expressed with the catalytically active subunit of mouse protein kinase A (PKA) encoded in a separate pACYC-PKA plasmid (chloramphenicol resistance), essentially as described earlier 26.
    pACYC-PKA
    suggested: None
    Crystallization and X-ray data collection: Crystallization was performed using 14-3-3σ surface entropy reduction mutants Clu1 or Clu3 (storage buffer 20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 2 mM MgCl2) mixed with either pS197 or pT205 phosphopeptide at a 1:5 protein:peptide molar ratio with the final protein concentration of 11.5 mg/ml.
    pS197
    suggested: None
    pT205
    suggested: None
    Software and Algorithms
    SentencesResources
    The Mw distributions were calculated in Astra 8.0 software (Wyatt Technology) using dn/dc=0.185.
    Astra
    suggested: (ASTRA, RRID:SCR_016255)
    To determine KD values, the binding curves were approximated using the quadratic equation 56 in Origin 9.0 (OriginLab Corporation, Northampton, MA, USA).
    OriginLab Corporation
    suggested: (Origin, RRID:SCR_014212)
    Peptide residues were manually built into difference electron density maps in Coot 59 and the overall structures were refined using Buster 2.10.4 60, which used NCS information (with pruning), TLS and all-atom individual isotropic B-factor restrained refinement.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Buster
    suggested: (BUSTER, RRID:SCR_015653)
    The refined structures were validated using the comprehensive validation algorithm in Phenix 1.10 62.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Structural illustrations were prepared using PyMOL 2.20 (Schrodinger, Inc.).
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    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.

    About SciScore

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  4. Version published to 10.1101/2021.12.23.474009v2 on bioRxiv
  5. Version published to 10.1101/2021.12.23.474009v1 on bioRxiv