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A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system

Nature Structural & Molecular Biology volume 16, pages 468–476 (2009)Cite this article

Abstract

The type III secretion system (T3SS) is a macromolecular 'injectisome' that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is composed of connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane secretin EscC and the inner membrane protein PrgH reveal the conservation of a modular fold among the three proteins that form the outer membrane and inner membrane rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized outer membrane and inner membrane rings characteristic of the T3SS. Using an integrated structural and experimental approach, we generated ring models for the periplasmic domain of EscC and placed them in the context of the assembled T3SS, providing evidence for direct interaction between the outer membrane and inner membrane ring components and an unprecedented span of the outer membrane secretin.

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Figure 1: Basal body of the T3SS.
Figure 2: EscC, PrgH and EscJ share a common fold for the assembly of the T3SS multiring basal body.
Figure 3: EscC21–174 and the T3SS-specific N-terminal secretin region within the T3SS needle complex.
Figure 4: EscC21–174 and the T3SS specific N-terminal secretin region within the T3SS needle complex.
Figure 5: Illustration of conformational sampling during protein-protein docking simulations.
Figure 6: Illustration of the evaluation process of the ring models.

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Acknowledgements

We thank the staff at the Advanced Light Source beamlines 8.2.2 and 8.3.1 for data collection, time and assistance. C.K.Y. thanks the Natural Science and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research (MSFHR). I.A. is supported by the Knut and Alice Wallenberg foundation. S.S. thanks the Natural Sciences and Engineering Research Council of Canada for postdoctoral funding. N.C.J.S. and B.B.F. thank the Howard Hughes International Scholar program and the Canadian Institutes of Health Research (CIHR) for funding. N.C.J.S. also thanks the MSFHR and Canadian Foundation for Innovation (CFI) for infrastructure funding support. This work was supported by the US National Institutes of Health (NIH) grant P20 GM076222 to D.B. and by NIH grants U54 AI05741 and RO1 AI030479 to S.I.M. We thank T. Walz (Harvard Medical School) for providing resources for the EM measurements, E.E. Galyov (University of Leicester) for providing the SipA, SipB and SipC antibodies and V. Koronakis (University of Cambridge) for providing the InvG antibody. We thank H. Felise, S. Schafer and D. Goodlett for technical advice and assistance.

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  1. Calvin K Yip & Tyler G Kimbrough

    Present address: Present addresses: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA (C.K.Y.); Department of Otolaryngology, University of Minnesota, Minneapolis, Minnesota, USA (T.G.K.).,

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada

    Thomas Spreter, Calvin K Yip, Marija Vuckovic, Angel C Yu & Natalie C J Strynadka

  2. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Sarah Sanowar, Richard A Pfuetzner & Samuel I Miller

  3. Department of Biochemistry, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Ingemar André & David Baker

  4. Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA

    Tyler G Kimbrough

  5. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Wanyin Deng & B Brett Finlay

  6. Department of Microbiology, University of Washington, Seattle, Washington, USA

    Samuel I Miller

  7. Department of Medicine, University of Washington, Seattle, Washington, USA

    Samuel I Miller

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Contributions

C.K.Y., M.V. and A.C.Y. cloned, purified and crystallized the EscC and PrgH constructs, and C.K.Y. determined the crystal structures; molecular modeling was carried out by I.A., and the models were analyzed, interpreted and docked into the EM maps by T.S. and I.A; S.S. did the biochemical characterization in S. typhimurium, and W.D. performed the experiments in EPEC; T.S. established the purification protocol for InvG; T.S. and N.C.J.S. wrote the paper; I.A. prepared Figure 5 and the computational methods; S.S. prepared Figure 4c and Supplementary Figure 3; W.D. prepared Figure 4b and Supplementary Figure 4; all authors discussed the results and commented on the manuscript; D.B. supervised all computational analysis; S.I.M. and B.B.F. provided resources for the biochemical characterizations in S. typhimurium and EPEC, respectively; N.C.J.S. provided resources for all data other than Figures 4b,c and 5 and Supplementary Figures 3 and 4.

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Correspondence to Natalie C J Strynadka.

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Spreter, T., Yip, C., Sanowar, S. et al. A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat Struct Mol Biol 16, 468–476 (2009). https://doi.org/10.1038/nsmb.1603

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