Crystal structures of SARS-CoV-2 ADP-ribose phosphatase: from the apo form to ligand complexes

  1. Karolina Michalska
  2. Youngchang Kim
  3. Robert Jedrzejczak
  4. Natalia I. Maltseva
  5. Lucy Stols
  6. Michael Endres
  7. Andrzej Joachimiak

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Abstract

Among 15 nonstructural proteins (Nsps), the newly emerging Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) encodes a large, multidomain Nsp3. One of its units is the ADP-ribose phosphatase domain (ADRP; also known as the macrodomain, MacroD), which is believed to interfere with the host immune response. Such a function appears to be linked to the ability of the protein to remove ADP-ribose from ADP-ribosylated proteins and RNA, yet the precise role and molecular targets of the enzyme remain unknown. Here, five high-resolution (1.07–2.01 Å) crystal structures corresponding to the apo form of the protein and its complexes with 2-( N -morpholino)ethanesulfonic acid (MES), AMP and ADP-ribose have been determined. The protein is shown to undergo conformational changes to adapt to the ligand in the manner previously observed in close homologues from other viruses. A conserved water molecule is also identified that may participate in hydrolysis. This work builds foundations for future structure-based research on ADRP, including the search for potential antiviral therapeutics.

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  1. Version published to 10.1107/s2052252520009653
  2. ScreenIT

    SciScore for 10.1101/2020.05.14.096081: (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

    Experimental Models: Organisms/Strains
    SentencesResources
    The residues that have not been modeled due to the lack of interpretable electron density include: Gly1-Glu2 and Glu170 in chains A and B for ADRP/APO1, Gly1-Glu2-Val3, Leu169-Glu170 in chain A and Gly1-Glu2, Glu170 in chain B for ADRP/ADPr, Gly1-Glu2 in chain A and Gly1-Glu2, Glu170 in chain B for ADRP/AMP, Gly1-Glu2-Val3 and Glu170 for ADRP/MES, and Gly1-Glu2 for ADRP/APO2.
    Gly1-Glu2
    suggested: None
    Software and Algorithms
    SentencesResources
    The protein solution was concentrated on 10 kDa MWCO filter (Amicon-Millipore) and further purified on size exclusion column Superdex 200 in lysis buffer where β-mercaptoethanol was replaced with 1 mM TCEP.
    Amicon-Millipore
    suggested: None
    Intensities were converted to structure factor amplitudes in the truncate program (French & Wilson, 1978, Padilla & Yeates, 2003) from the CCP4 package (Winn et al., 2011).
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    In all cases, the initial solution was manually adjusted using COOT (Emsley & Cowtan, 2004) and then iteratively refined using Coot, Phenix (Adams et al., 2010) and Refmac (Murshudov et al., 1997, Winn et al., 2011).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Throughout the refinement, the same 5% of reflections were kept out throughout from the refinement (in both REFMAC and PHENIX refinements).
    REFMAC
    suggested: (Refmac, RRID:SCR_014225)
    The stereochemistry of the structure was checked with MolProbity (Davis et al., 2007) PROCHECK (Laskowski et al., 1993) and the Ramachandran plot and validated with the PDB validation server.
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    A more diverged picture is observed in distant homologs from H-CoV-229E, IBV, and FIPV (Fig. 6), mainly in the A and R1 sites, with caveat that the distal ribose in H-CoV-229E ADRP complex has wrong stereochemistry. In these homologs, we observe sequence variation in the F156 position, which is replaced by other hydrophobic residues. The adenine ring is significantly shifted with respect to SARS-CoV-2 ADRP. The interaction between N1 of adenine and D22 equivalent is lost, even though the latter amino acid is conserved in the 3-dimensional context (in IBV, D20 does not overlap in the primary sequence). The distal ribose is better anchored in place – hydrogen bonds link it either to the glutamate residue (E156 in H-CoV-229E, E191 in FIPV) that substitutes L160, or serine in position of V155 (S160 in IBV). Another notable difference is observed in the R2 site, where equivalents of I131 in H-CoV-229E and IBV proteins adopt outlier rotamers, yet the electron density maps allow to model more favorable conformations seen in our structure. In these two models, the proximal ribose adopts α configuration on the anomeric carbon atom (Fig. 6). Such a state, with partial occupancy was also reported for one of the SARS-CoV/ADPr complexes (Egloff et al., 2006) and is linked to the alternative, apo-like conformation of G47-G48. The α configuration most likely illustrates the geometry of the putative substrate, as only then the hydroxyl group is exposed to the solvent, providing room for the ...

    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.

    About SciScore

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  3. Version published to 10.1101/2020.05.14.096081v2 on bioRxiv
  4. Version published to 10.1101/2020.05.14.096081v1 on bioRxiv