The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and x-ray diffraction at room temperature

  1. Galen J. Correy
  2. Daniel W. Kneller
  3. Gwyndalyn Phillips
  4. Swati Pant
  5. Silvia Russi
  6. Aina E. Cohen
  7. George Meigs
  8. James M. Holton
  9. Stefan Gahbauer
  10. Michael C. Thompson
  11. Alan Ashworth
  12. Leighton Coates
  13. Andrey Kovalevsky
  14. Flora Meilleur
  15. James S. Fraser

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Abstract

The nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Mac1) removes adenosine diphosphate (ADP) ribosylation posttranslational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the coronavirus disease 2019 pandemic. Here, we determined neutron and x-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose–bound states. We characterize extensive solvation in the Mac1 active site and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase the potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a reevaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

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  1. Version published to 10.1126/sciadv.abo5083
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    SciScore for 10.1101/2022.02.07.479477: (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
    P43 crystals: Protein expression and purification: The gene encoding SARS-CoV-2 NSP3 Mac1 (residues 3-169) was cloned into a pET-22b(+) expression plasmid with a TEV-cleavable N-terminal 6-His tag (Genscript).
    pET-22b(+)
    suggested: RRID:Addgene_12651)
    crystals: Protein expression and purification: The gene encoding SARS-CoV-2 2-170 NSP3 Mac1 was cloned into a pET-11a plasmid (Bio Basic) and transformed into E. coli (BL21-DE3).
    pET-11a
    suggested: RRID:Addgene_154201)
    Software and Algorithms
    SentencesResources
    After five macrocycles of refinement, the 2mFO-DFC (unfilled) and the mFO-DFC NSL density maps were inspected in Coot and modifications made to the coordinates.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Phases were obtained by molecular replacement with Phaser using the same search model as the lower resolution X-ray experiment.
    Phaser
    suggested: (Phaser, RRID:SCR_014219)
    The number of contacts made by water molecules was defined as the number of protein nitrogen or oxygen atoms within 3.5 Å of a water, with distances calculated using PyMOL.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One caveat to using the neutron-determined orientations is that only orientations of highly ordered water molecules could be determined with high confidence (Fig. 5D/E). Fortunately, these waters are most frequently involved in bridging interactions. Simulation-based methods might yield an improved model of the orientations of disordered water molecules (65). We found that the conformational ensemble of Mac1 was unusually robust to temperature and pH perturbation in the crystal. Room temperature crystallography can reveal low occupancy, functionally relevant conformational states of proteins (51, 66–68). The 1.1 Å room temperature structure of Mac1 reported here, in combination with the previously reported 0.85 Å structure determined using the same crystal form at 100 K (14), allowed a detailed analysis of temperature dependent changes in structure and function. Overall, the structure and flexibility at 100 and 293 K were remarkably similar, indicating that unlike several recently reported examples (68–70), Mac1 displays minimal temperature dependent structural changes in crystallo (Fig. 4C). Similarly, assuming that the crystals have equilibrated to the target pH in the ∼4 hour soaks conducted here (71), the pH shift experiments revealed that the Mac1 active site residues and water networks are remarkably robust (Fig. 6I/J), and this is matched by the invariance in ADPr binding across the entire pH range (Fig. 6G/H). One caveat to the pH-shift experiments is the pH-dependent...

    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.

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  3. Version published to 10.1101/2022.02.07.479477v1 on bioRxiv