The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (M pro )

  1. Ali Ebrahim
  2. Blake T. Riley
  3. Desigan Kumaran
  4. Babak Andi
  5. Martin R. Fuchs
  6. Sean McSweeney
  7. Daniel A. Keedy

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Abstract

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or M pro , is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of M pro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded M pro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for M pro , including mobile solvent interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19 and combat future coronavirus pandemics.

Synopsis

X-ray crystallography at variable temperature for SARS-CoV-2 M pro reveals a complex conformational landscape, including mobile solvent at the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and an intramolecular network bridging the active site and dimer interface.

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  1. Version published to 10.1101/2021.05.03.437411v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.05.03.437411: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationNext, a phenix.ensemble_refinement grid search was performed by repeating the simulation with four values of pTLS (1.0, 0.9, 0.8, 0.6) and three values of wxray_coupled_tbath_offset (10, 5, 2.5), and using a random_seed value of 2679941.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    Briefly, the codon-optimized synthetic gene of full-length Mpro from SARS-CoV-2 was cloned into the pET29b vector.
    pET29b
    suggested: RRID:Addgene_128795)
    Software and Algorithms
    SentencesResources
    Molecular replacement for each dataset was performed via Phaser-MR from the Phenix software suite, using PDB ID 6YB7 as a search model.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Geometric and protein statistics of the final models were evaluated via MolProbity 53,54 and (https://smb.slac.stanford.edu/jcsg/QCI).
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    For solvent content analysis, rwcontents v7.1.009 from the CCP4 suite55 was used.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)

    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.

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  3. Version published to 10.1101/2021.05.03.437411v1 on bioRxiv