Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography

  1. Daniel W. Kneller
  2. Gwyndalyn Phillips
  3. Kevin L. Weiss
  4. Swati Pant
  5. Qiu Zhang
  6. Hugh M. O’Neill
  7. Leighton Coates
  8. Andrey Kovalevsky

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Abstract

The main protease (3CL M pro ) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL M pro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL M pro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature – the critical information for structure-assisted and computational drug design.

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    SciScore for 10.1101/2020.09.22.308668: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Diffraction data were then reduced and scaled using the Aimless53 program from the CCP4 suite54; molecular replacement using PDB code 6WQF25 was then performed with Molrep54 from the CCP4 program suite.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    The geometry of the final model was then carefully checked with Molprobity.
    Molprobity
    suggested: (MolProbity, RRID:SCR_014226)
    Joint X-ray/neutron refinement: The joint X-ray/neutron refinement of ligand-free 3CL Mpro was performed using nCNS58,59 and the structure was manipulated in Coot.
    Coot
    suggested: (Coot, RRID:SCR_014222)

    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.

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  2. Version published to 10.1101/2020.09.22.308668v1 on bioRxiv