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.

Reviewing Editor

  1. Wade Harper, Harvard Medical School, United States

Version history

  1. Received: April 26, 2020
  2. Accepted: November 12, 2020
  3. Accepted Manuscript published: November 13, 2020 (version 1)
  4. Version of Record published: November 26, 2020 (version 2)

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

  • 2,809
    views
  • 342
    downloads
  • 24
    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
    2. Structural Biology and Molecular Biophysics
    Thomas RM Germe, Natassja G Bush ... Anthony Maxwell
    Research Article

    DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. DNA gyrase can loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion from a positive (+1) to a negative (–1) supercoil, thereby introducing negative supercoiling into the bacterial genome by steps of 2, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper, we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface ‘swapping’ (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. Interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface swapping. This swapping does not require ATP, occurs in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed ‘swivelling’ mechanism for DNA gyrase (Gubaev et al., 2016).

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.