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Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases

Nature Structural & Molecular Biology volume 15, pages 1293–1301 (2008)Cite this article

Abstract

IpaH proteins are E3 ubiquitin ligases delivered by the type III secretion apparatus into host cells upon infection of humans by the Gram-negative pathogen Shigella flexneri. These proteins comprise a variable leucine-rich repeat–containing N-terminal domain and a conserved C-terminal domain harboring an invariant cysteine residue that is crucial for activity. IpaH homologs are encoded by diverse animal and plant pathogens. Here we demonstrate that the IpaH C-terminal domain carries the catalytic activity for ubiquitin transfer and that the N-terminal domain carries the substrate specificity. The structure of the IpaH C-terminal domain, determined to 2.65-Å resolution, represents an all-helical fold bearing no resemblance to previously defined E3 ubiquitin ligases. The conserved and essential cysteine residue lies on a flexible, surface-exposed loop surrounded by conserved acidic residues, two of which are crucial for IpaH activity.

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Figure 1: The C-terminal domain of IpaH proteins shows E3 ubiquitin-ligase activity.
Figure 2: E3 ubiquitin ligase activity of full-length IpaH proteins.
Figure 3: Overall structure of the IpaH1.4 C-terminal domain.
Figure 4: Multiple sequence alignment of IpaH homologs.
Figure 5: Environment of the conserved cysteine residue.
Figure 6: Functional analysis of conserved residues.
Figure 7: IpaH interactions with E2 conjugating enzymes.
Figure 8: Structures of different classes of E3 ligases in which a cysteine residue is essential for activity.

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Acknowledgements

We wish to thank the staff at the Argonne National Laboratory beam line 19-ID for assistance with data collection and A. Edwards for critical reading of the manuscript. We also wish to thank S. Dhe-Paganon and G. Avvakumov at the Structural Genomics Consortium, Toronto, for providing the collection of expression constructs for human E2-conjugating enzymes. We thank D. Briant for insight to E3 ubiquitination assays. This work was supported by US National Institutes of Health Grants GM62414-01, by the Ontario Research and Development Challenge Fund and by a grant from the Canadian Institutes of Health Research Grant. M.T. is supported by grants from the Canadian Institutes of Health Research (MT012466 and MOP-57795), the National Cancer Institute of Canada, the Royal Society and the Scottish Universities Life Sciences Alliance.

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Author notes

  1. Alexander U Singer and John R Rohde: These authors contributed equally to this work.

Authors and Affiliations

  1. Banting and Best Department for Medical Research, Ontario Centre for Structural Proteomics, Midwest Centre for Structural Proteomics, University of Toronto, C.H. Best Institute, Room 24, 112 College Street, Toronto, M5G 1L5, Ontario, Canada

    Alexander U Singer, Tatiana Skarina, Olga Kagan, Rosa DiLeo & Alexei Savchenko

  2. Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Room 989, 600 University Avenue, Toronto, M5G 1X5, Ontario, Canada

    John R Rohde & Mike Tyers

  3. Ontario Cancer Institute, Toronto, MBRC 5th floor, 200 Elizabeth Street, M5G 2C4, Ontario, Canada

    Robert Lam & Nickolay Y Chirgadze

  4. Department of Pharmacology and Toxicology, University of Toronto, 1 King's College Circle, M5S 1A8, Ontario, Canada

    Nickolay Y Chirgadze

  5. Midwest Center for Structural Genomics and Structural Biology Center, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, 60439, Illinois, USA

    Marianne E Cuff & Andrzej Joachimiak

  6. Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur, 28 Rue du Docteur Roux, Paris, 75724, Cedex 15, France

    Philippe J Sansonetti & Claude Parsot

  7. Unité INSERM, U786, 28 Rue du Docteur Roux, Paris, 75724, Cedex 15, France

    Philippe J Sansonetti & Claude Parsot

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Contributions

A.U.S. and R.L. determined the structure of the IpaH-CTD and contributed the data for Figures 2, 4 and 6 and Supplementary Figure 1. J.R.R. contributed the data for Figures 1, 2 and 6. T.S. and O.K. purified and crystallized IpaH-CTD. T.S. also contributed the data for Figures 1, 2, 6 and 7. R.D. subcloned most of the constructs. M.E.C. helped with diffraction data collection. A.U.S., J.R.R., N.Y.C., A.J., R.L., M.T., P.J.S., C.P. and A.S. designed experiments, analyzed data and prepared the manuscript.

Corresponding author

Correspondence to Alexei Savchenko.

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Singer, A., Rohde, J., Lam, R. et al. Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases. Nat Struct Mol Biol 15, 1293–1301 (2008). https://doi.org/10.1038/nsmb.1511

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