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Structure of the cholera toxin secretion channel in its closed state

Nature Structural & Molecular Biology volume 17, pages 1226–1232 (2010)Cite this article

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Abstract

The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.

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Figure 1: Purification and cryo-EM of the cholera toxin secretion channel VcGspD.
Figure 2: Three-dimensional electron cryo-EM reconstruction of VcGspD.
Figure 3: The secretin architecture is conserved in different secretion systems.
Figure 4: The periplasmic GspD domain contains a conserved N3 constriction and binds to the T2SS exoprotein and pseudopilus tip complex.
Figure 5: Piston-driven mechanism for protein secretion.

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Acknowledgements

We thank the Murdock Charitable Trust and the Washington Research Foundation for generous support of our cryo-EM facility. We are grateful to J. Sun, M. Gonen, B. Vollmar and S. Turley for contributions to the earlier stages of this work; M. Bagdasarian (Michigan State University) for a VcGspD-containing plasmid; and J. DelaRosa for assistance with protein preparation. We thank A. J. Merz for helpful discussions. We thank N. Korotkova and P. Wallace for discussion of SPR experiments. Part of this work was conducted at the University of Washington NanoTech User Facility, a member of the US National Science Foundation (NSF) National Nanotechnology Infrastructure Network (NNIN). This research is supported by the US National Institutes of Health grant AI34501. The Gonen laboratory is supported in part by the Howard Hughes Medical Institute Early Career Scientist program.

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  1. Steve L Reichow and Konstantin V Korotkov: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Steve L Reichow, Konstantin V Korotkov, Wim G J Hol & Tamir Gonen

  2. Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Tamir Gonen

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Contributions

T.G. and W.G.J.H. designed the research; K.V.K. cloned, expressed and purified protein samples, constructed the molecular models of the periplasmic rings of VcGspD and of the pseudopilus and performed SPR experiments; S.L.R. collected and processed the cryo-EM data and prepared all figures; all authors wrote the manuscript.

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Correspondence to Wim G J Hol or Tamir Gonen.

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Reichow, S., Korotkov, K., Hol, W. et al. Structure of the cholera toxin secretion channel in its closed state. Nat Struct Mol Biol 17, 1226–1232 (2010). https://doi.org/10.1038/nsmb.1910

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