Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection

  1. Donghyuk Shin
  2. Anshu Bhattacharya
  3. Yi-Lin Cheng
  4. Marta Campos Alonso
  5. Ahmad Reza Mehdipour
  6. Gerbrand J van der Heden van Noort
  7. Huib Ovaa
  8. Gerhard Hummer
  9. Ivan Dikic  Is a corresponding author
  1. Institute of Biochemistry II, Germany
  2. Max Planck Institute of Biophysics, Germany
  3. Leiden University Medical Center, Netherlands
  4. Leiden University Medical Centre, Netherlands

Abstract

Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two Legionella OTU-like deubiquitinases (LOT; LotB (Lpg1621/Ceg23) and LotC (Lpg2529)). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the Legionella OTU-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-deubiquitinases. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU deubiquitinases and indicate distinct roles in host-pathogen interactions.

Data availability

Diffraction data have been deposited in PDB under the accession code 6YK8.

The following data sets were generated

Article and author information

Author details

  1. Donghyuk Shin

    Faculty of Medicine, Institute of Biochemistry II, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8272-6133
  2. Anshu Bhattacharya

    Faculty of Medicine, Institute of Biochemistry II, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7383-3594
  3. Yi-Lin Cheng

    Faculty of Medicine, Institute of Biochemistry II, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  4. Marta Campos Alonso

    Faculty of Medicine, Institute of Biochemistry II, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  5. Ahmad Reza Mehdipour

    Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt, Germany
    Competing interests
    No competing interests declared.
  6. Gerbrand J van der Heden van Noort

    Cell and Chemical Biology, Leiden University Medical Center, Leiden, Netherlands
    Competing interests
    No competing interests declared.
  7. Huib Ovaa

    Cell and Chemical Biology, Leiden University Medical Centre, Leiden, Netherlands
    Competing interests
    No competing interests declared.
  8. Gerhard Hummer

    Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7768-746X
  9. Ivan Dikic

    Faculty of Medicine, Institute of Biochemistry II, Frankfurt am Main, Germany
    For correspondence
    dikic@biochem2.uni-frankfurt.de
    Competing interests
    Ivan Dikic, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8156-9511

Funding

H2020 European Research Council (742720)

  • Donghyuk Shin
  • Anshu Bhattacharya
  • Yi-Lin Cheng
  • Marta Campos Alonso

Deutsche Forschungsgemeinschaft (ID 259139777)

  • Ahmad Reza Mehdipour
  • Gerhard Hummer
  • Ivan Dikic

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2020, Shin et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,910
    views
  • 348
    downloads
  • 27
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Donghyuk Shin
  2. Anshu Bhattacharya
  3. Yi-Lin Cheng
  4. Marta Campos Alonso
  5. Ahmad Reza Mehdipour
  6. Gerbrand J van der Heden van Noort
  7. Huib Ovaa
  8. Gerhard Hummer
  9. Ivan Dikic
(2020)
Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection
eLife 9:e58277.
https://doi.org/10.7554/eLife.58277

Share this article

https://doi.org/10.7554/eLife.58277

Further reading

    1. Biochemistry and Chemical Biology
    Bernd K Gilsbach, Franz Y Ho ... Christian Johannes Gloeckner
    Research Article

    The Parkinson’s disease (PD)-linked protein Leucine-Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis–Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lies within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM and the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer–dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity, suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together, our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase-dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Katherine A Senn, Karli A Lipinski ... Aaron A Hoskins
    Research Article

    Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.