1. Structural Biology and Molecular Biophysics

Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

  1. Alan Sulpizio
  2. Marena E Minelli
  3. Min Wan
  4. Paul D Burrowes
  5. Xiaochun Wu
  6. Ethan J Sanford
  7. Jung-Ho Shin
  8. Byron C Williams
  9. Michael L Goldberg
  10. Marcus B Smolka
  11. Yuxin Mao  Is a corresponding author
  1. Cornell University, United States
https://doi.org/10.7554/eLife.51162
Share this article
Cite this article
  1. Alan Sulpizio
  2. Marena E Minelli
  3. Min Wan
  4. Paul D Burrowes
  5. Xiaochun Wu
  6. Ethan J Sanford
  7. Jung-Ho Shin
  8. Byron C Williams
  9. Michael L Goldberg
  10. Marcus B Smolka
  11. Yuxin Mao
(2019)
Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ
eLife 8:e51162.
https://doi.org/10.7554/eLife.51162
  2. Download BibTeX
  3. Download .RIS

Abstract

Pseudokinases are considered to be the inactive counterparts of conventional protein kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the eukaryotic Ca2+-binding regulator, calmodulin (CaM). The structure reveals that SidJ contains a protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro biochemical analyses demonstrate that SidJ modifies another Legionella effector SdeA, an unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some pseudokinases may possess ATP-dependent activities other than conventional phosphorylation.

Data availability

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

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Alan Sulpizio

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  2. Marena E Minelli

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  3. Min Wan

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Paul D Burrowes

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Xiaochun Wu

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ethan J Sanford

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Jung-Ho Shin

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  8. Byron C Williams

    Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  9. Michael L Goldberg

    Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0200-0277

  10. Marcus B Smolka

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    Competing interests
    The authors declare that no competing interests exist.

  11. Yuxin Mao

    Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, United States
    For correspondence
    ym253@cornell.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5064-1397

Funding

National Institute for Health Research (5R01GM116964)

  • Yuxin Mao

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

Copyright

© 2019, Sulpizio 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

  • 2,553
    views
  • 319
    downloads
  • 63
    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. Alan Sulpizio
  2. Marena E Minelli
  3. Min Wan
  4. Paul D Burrowes
  5. Xiaochun Wu
  6. Ethan J Sanford
  7. Jung-Ho Shin
  8. Byron C Williams
  9. Michael L Goldberg
  10. Marcus B Smolka
  11. Yuxin Mao
(2019)
Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ
eLife 8:e51162.
https://doi.org/10.7554/eLife.51162

Share this article

https://doi.org/10.7554/eLife.51162

Categories and tags

Further reading

    1. Structural Biology and Molecular Biophysics

    Molecular determinants of Neu5Ac binding to a tripartite ATP independent periplasmic (TRAP) transporter

    Parveen Goyal, KanagaVijayan Dhanabalan ... Subramanian Ramaswamy
    Research Advance

    N -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+ binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.

    1. Computational and Systems Biology
    2. Structural Biology and Molecular Biophysics

    Discovery of a heparan sulfate binding domain in monkeypox virus H3 as an anti-poxviral drug target combining AI and MD simulations

    Bin Zheng, Meimei Duan ... Peng Zheng
    Research Article

    Viral adhesion to host cells is a critical step in infection for many viruses, including monkeypox virus (MPXV). In MPXV, the H3 protein mediates viral adhesion through its interaction with heparan sulfate (HS), yet the structural details of this interaction have remained elusive. Using AI-based structural prediction tools and molecular dynamics (MD) simulations, we identified a novel, positively charged α-helical domain in H3 that is essential for HS binding. This conserved domain, found across orthopoxviruses, was experimentally validated and shown to be critical for viral adhesion, making it an ideal target for antiviral drug development. Targeting this domain, we designed a protein inhibitor, which disrupted the H3-HS interaction, inhibited viral infection in vitro and viral replication in vivo, offering a promising antiviral candidate. Our findings reveal a novel therapeutic target of MPXV, demonstrating the potential of combination of AI-driven methods and MD simulations to accelerate antiviral drug discovery.

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics

    Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging

    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.