Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

  1. Pawel M. Wydorski
  2. Jerzy Osipiuk
  3. Benjamin T. Lanham
  4. Christine Tesar
  5. Michael Endres
  6. Elizabeth Engle
  7. Robert Jedrzejczak
  8. Vishruth Mullapudi
  9. Karolina Michalska
  10. Krzysztof Fidelis
  11. David Fushman
  12. Andrzej Joachimiak
  13. Lukasz A. Joachimiak

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Abstract

The Papain-like protease (PLpro) is a domain of a multi-functional, non-structural protein 3 of coronaviruses. PLpro cleaves viral polyproteins and posttranslational conjugates with poly-ubiquitin and protective ISG15, composed of two ubiquitin-like (UBL) domains. Across coronaviruses, PLpro showed divergent selectivity for recognition and cleavage of posttranslational conjugates despite sequence conservation. We show that SARS-CoV-2 PLpro binds human ISG15 and K48-linked di-ubiquitin (K48-Ub 2 ) with nanomolar affinity and detect alternate weaker-binding modes. Crystal structures of untethered PLpro complexes with ISG15 and K48-Ub 2 combined with solution NMR and cross-linking mass spectrometry revealed how the two domains of ISG15 or K48-Ub 2 are differently utilized in interactions with PLpro. Analysis of protein interface energetics predicted differential binding stabilities of the two UBL/Ub domains that were validated experimentally. We emphasize how substrate recognition can be tuned to cleave specifically ISG15 or K48-Ub 2 modifications while retaining capacity to cleave mono-Ub conjugates. These results highlight alternative druggable surfaces that would inhibit PLpro function.

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  1. Version published to 10.1101/2021.09.15.460543v3 on bioRxiv
  2. Version published to 10.1101/2021.09.15.460543v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.09.15.460543: (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

    Experimental Models: Cell Lines
    SentencesResources
    MCSG1, MCSG2, MCSG3, and MCSG4 (Anatrace), Index (Hampton Research) and Wizard 1&2 (Jena Bioscience) screens were used at 16 °C.
    MCSG1
    suggested: None
    For the PLpro-C111S/Ub2-K48R,D77 complex (11 mg/ml), crystals appeared in MCSG-2 F11 and were improved in hanging drops with a protein-to-matrix ratio of 2:1 in 0.2 M di-sodium tartrate, 15 % (w/v) PEG3350 after seeding with 1/10 volume of PLpro-C111S/Ub2-K48R,D77 microcrystals.
    MCSG-2 F11
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    All cross-linking reactions were quenched with 172 mM (4 times excess) Ammonium Bicarbonate (AB) for 30 minutes at 37 °C while shaking at 350 rpm.
    AB
    suggested: None
    Recombinant DNA
    SentencesResources
    Human ISG15 gene was also synthesised and cloned directly into pMCSG53 vector.
    pMCSG53
    suggested: RRID:Addgene_167255)
    Unlabeled Ub variants were expressed in BL12(DE3) E.coli cells containing a helper pJY2 plasmid and purified as described elsewhere27.
    pJY2
    suggested: None
    Software and Algorithms
    SentencesResources
    PALMIST and GUSSI software is freely available for academic users on UTSW MBR core’s website (https://www.utsouthwestern.edu/labs/mbr/software/).
    GUSSI
    suggested: (GUSSI, RRID:SCR_014962)
    Intensities were converted to structure factor amplitudes in the truncate program33 from the CCP4 package34.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    Several rounds of manual adjustments of structure models using COOT39 and refinements with REFMAC program38 from CCP4 suite34 were done.
    REFMAC
    suggested: (Refmac, RRID:SCR_014225)
    The stereochemistry of the structure was validated with PHENIX suite40 incorporating MOLPROBITY41 tools.
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    The data were processed using Topspin (Bruker) and analyzed using Sparky 3.19042.
    Sparky
    suggested: (Sparky , RRID:SCR_014228)
    Selection of common interface residues was carried out in PyMOL 2.4.1.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    These simulations were performed using BioHPC computing cluster at UT Southwestern Medical Center.
    BioHPC
    suggested: None
    The plots were made using GraphPad Prism 8 and mapped onto the protease structure using PyMOL 2.4.1.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The relax protocol and Flex_ΔΔG used Rosetta v3.13 and v3.12, respectively.
    Rosetta
    suggested: (Rosetta, RRID:SCR_015701)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The original structure of PLproCoV-1 bound to K48-linked Ub2 revealed both Ubs bound to the protease through binding sites present in both proximal and distal Ubs with minor caveats of the linkage not being hydrolysable and the C-terminal tail covalently linked to the protease. There remains no structure of the ISG15 bound to PLproCoV-1 suggesting that the dramatically reduced affinity must impact binding both proximal and distal binding UBL domains of ISG15. Our new structure of PLproCoV-2 bound to human ISG15 as well as the previously determined structure of PLproCoV-2 bound to mISG15 reveal a dual UBL domain recognition binding mode despite surprising sequence variation between the mouse and human forms of ISG15, in particular the substrate binding interface (Fig. 1b). Our in silico alanine scan of the interfaces uncovered which residues may play central roles in stabilizing the ISG15 binding mode for PLproCoV-2 and implicated V66 as being important for stabilizing the distal UBL domain of ISG15. Furthermore, our evolutionary analysis of SARS-CoV-2 variants highlights sequence variation at key sites suggesting the PLpro variants exist that may have altered substrate specificity with unknown disease outcomes. Impact of PLpro sequence drift on substrate specificity: Much of the focus on understanding how sequence variation impacts pathogenicity, infectivity and virulence of SARS-CoV-2 has been centered on sequence changes in surface proteins such as the receptor-binding doma...

    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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  4. Version published to 10.1101/2021.09.15.460543v1 on bioRxiv