Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

  1. Mikhail A. Hameedi
  2. Erica T. Prates
  3. Michael R. Garvin
  4. Irimpan I. Mathews
  5. B. Kirtley Amos
  6. Omar Demerdash
  7. Mark Bechthold
  8. Mamta Iyer
  9. Simin Rahighi
  10. Daniel W. Kneller
  11. Andrey Kovalevsky
  12. Stephan Irle
  13. Van-Quan Vuong
  14. Julie C. Mitchell
  15. Audrey Labbe
  16. Stephanie Galanie
  17. Soichi Wakatsuki
  18. Daniel Jacobson

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Abstract

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like protease (3CLpro) can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.50 Å resolution crystal structure of 3CLpro C145S bound to NEMO 226–234 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for, in the pathology of COVID-19.

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  1. Version published to 10.1038/s41467-022-32922-9
  2. ScreenIT

    SciScore for 10.1101/2021.11.11.468228: (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
    Enzymatic assays: NEMO peptide expression and purification: The constructs of Human NEMO (residues 215-247) and mouse NEMO (residues 221-250) cloned into pGEX-6p-1 vector were transformed into BL21 (DE3) cells and selected using ampicillin-enriched LB media.
    pGEX-6p-1
    suggested: None
    3CLpro expression and purification: 3CLpro WT enzyme for assays was prepared independently from a clone of the SARS-CoV-2 NSP5 gene in pD451-SR (Atum, Newark, CA) according to published procedure 6.
    pD451-SR
    suggested: None
    Bands were visualized with BullDog Bio Acquastain. 5.2. Crystallography: 3CLpro WT expression and purification: BL21(DE3) cells were transformed with pMCSG53 pDNA containing a 3CLpro WT insert with an autoprocessing-sensitive N-terminal Maltose Binding Protein (MBP) tag and a PreScission protease-sensitive C-terminal His6 tag (provided by Andrzej Joachimiak).
    pMCSG53 pDNA
    suggested: None
    Software and Algorithms
    SentencesResources
    Iterative refinement was performed manually in Coot 51 and REFMAC 52.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    All bonds of the peptide were kept rigid as the goal was to preserve the initial conformation and compute the binding free energy using the AutoDock Vina scoring function.
    AutoDock
    suggested: (AutoDock, RRID:SCR_012746)

    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: 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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  3. Version published to 10.1101/2021.11.11.468228v1 on bioRxiv