Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

  1. Helder Veras Ribeiro Filho
  2. Gabriel Ernesto Jara
  3. Fernanda Aparecida Heleno Batista
  4. Gabriel Ravanhani Schleder
  5. Celisa Caldana Tonoli
  6. Adriana Santos Soprano
  7. Samuel Leite Guimarães
  8. Antonio Carlos Borges
  9. Alexandre Cassago
  10. Marcio Chaim Bajgelman
  11. Rafael Elias Marques
  12. Daniela Barreto Barbosa Trivella
  13. Kleber Gomes Franchini
  14. Ana Carolina Migliorini Figueira
  15. Celso Eduardo Benedetti
  16. Paulo Sergio Lopes de Oliveira

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Abstract

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N-(NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we showed that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that was consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.

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  1. Version published to 10.1101/2021.08.27.457964v2 on bioRxiv
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    SciScore for 10.1101/2021.08.27.457964: (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 N protein sequence (GenBank QIG56001.1) was amplified from cDNA samples using primers SC2-protN28182-F (5’-AGTCTTGTAGTGCGTTGTTCG-3’) and SC2-protN29566-R (5’-ATAGCCCATCTGCCTTGTGT-3’) and cloned into pGEM-T Easy (PROMEGA - USA), generating plasmid pGEM-SC2-N.
    pGEM-T Easy
    suggested: RRID:Addgene_80557)
    pGEM-SC2-N
    suggested: None
    The N sequence was reamplified from pGEM-SC2N with forward 5’-AACAAGCTAGCATGTCTGATAATGGACCCCAAAATCAG-3’ and reverse 5’-GGTCTGCGGCCGCTTAGGCCTGAGTTGAGTCAGCACTGCT-3’ primers and subcloned into the NheI/NotI sites of a pET28a-TEV vector carrying a 6xHis-tag and TEV protease cleavage site at the N-terminus.
    pGEM-SC2N
    suggested: None
    pET28a-TEV
    suggested: RRID:Addgene_168267)
    Software and Algorithms
    SentencesResources
    Then, the initial 3D map was refined in Imagic using an iterative process of angular reconstitution and class average rotation and translation alignment to 3D reprojections, achieving 3D resolution convergence.
    Imagic
    suggested: (IMAGIC, RRID:SCR_014447)
    CG molecular dynamics simulations: CG molecular dynamics simulations were performed using CafeMol 3.1 software52.
    CafeMol
    suggested: None
    An initial N protein dimer all-atom model was built in YASARA software53 using crystallographic structures of NTD monomer (PDB ID: 6VYO) and CTD dimer (PDB ID: 6WJI).
    YASARA
    suggested: (YASARA, RRID:SCR_017591)
    Tools for comparing CG simulations with experimental data: For comparing CG simulation radius of gyration with experimental data, we estimated the radius of gyration of the simulated systems with Bio3D package using an in-house R script54.
    Bio3D
    suggested: None
    The area of 2D reprojections was determined using ImageJ software56. 3D Atomic model fitting: From the previous independent simulation of the CG model of N protein dimer in complex with a 60-nt-long RNA were selected 100 structures with the lowest radius of gyration using only the structures domains (NTDs and CTDs).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The molecular interactions were described by CHARMM36 force field with CMAP corrections60.
    CMAP
    suggested: (CMAP, RRID:SCR_009034)
    All simulations were performed with NAMD 2.1361.
    NAMD
    suggested: (NAMD, RRID:SCR_014894)
    All the systems were described using Amber ff14SB force field68 and the topologies were generated using tLeap program from AmberTool2069.
    Amber
    suggested: (AMBER, RRID:SCR_016151)
    tLeap
    suggested: None
    The images of the C-terminal tail monomers and dimers were generated using pymol 2.3.0 (open-source build).
    pymol
    suggested: (PyMOL, RRID:SCR_000305)

    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 38. 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.

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

    About SciScore

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  3. Version published to 10.1101/2021.08.27.457964v1 on bioRxiv