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Protein refolding is required for assembly of the type three secretion needle

Nature Structural & Molecular Biology volume 17, pages 788–792 (2010)Cite this article

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Abstract

Pathogenic Gram-negative bacteria use a type three secretion system (TTSS) to deliver virulence factors into host cells. Although the order in which proteins incorporate into the growing TTSS is well described, the underlying assembly mechanisms are still unclear. Here we show that the TTSS needle protomer refolds spontaneously to extend the needle from the distal end. We developed a functional mutant of the needle protomer from Shigella flexneri and Salmonella typhimurium to study its assembly in vitro. We show that the protomer partially refolds from α-helix into β-strand conformation to form the TTSS needle. Reconstitution experiments show that needle growth does not require ATP. Thus, like the structurally related flagellar systems, the needle elongates by subunit polymerization at the distal end but requires protomer refolding. Our studies provide a starting point to understand the molecular assembly mechanisms and the structure of the TTSS at atomic level.

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Figure 1: Polymerization of a functional TTSS protomer.
Figure 2: In vitro assembly of TTSS needles.
Figure 3: Protomer refolding during assembly of the TTSS needle.
Figure 4: Assembly of the TTSS needle.

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Acknowledgements

We thank P. Jungblut and M. Schmid for help with MS analysis, V. Brinkmann and U. Abu Abed for help with electron microscopy sample preparation, U. Müller and S. Monaco for assistance in using beamlines, M. Luft for providing the opportunity of the FTIR measurements and for helpful discussions, B. Angerstein for help with ssNMR sample preparation, D. Lee for help in using Bruker spectrometers and B. Raupach, A. Zumsteg, F. Meissner, M. Lunelli and R. Kumar Lokareddy for their useful comments and critical reading of the manuscript. This work was supported by the Max-Planck-Society (to C. Griesinger and A.Z.) and by the Deutsche Forschungsgemeinschaft through an Emmy Noether stipend (LA 2705/1-1) to A.L.

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Author notes

  1. Ömer Poyraz, Karsten Seidel, Christian Ader & Marc Baldus

    Present address: Present addresses: BASF SE, Ludwigshafen, Germany (K.S.), Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands (C.A. and M.B.) and Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden (Ö.P.).,

  2. Ömer Poyraz, Holger Schmidt and Karsten Seidel: These authors contributed equally to this work.

Authors and Affiliations

  1. Max-Planck-Institute for Infection Biology, Cellular Microbiology, Berlin, Germany

    Ömer Poyraz, Hezi Tenenboim, Arturo Zychlinsky & Michael Kolbe

  2. Max-Planck-Institute for Biophysical Chemistry, NMR based Structural Biology, Göttingen, Germany

    Holger Schmidt, Karsten Seidel, Christian Ader, Marc Baldus, Adam Lange & Christian Griesinger

  3. BAM Federal Institute for Material Research and Testing, Berlin, Germany

    Friedmar Delissen & Andreas F Thünemann

  4. Max-Planck-Institute for Infection Biology, Core Facility Microscopy, Berlin, Germany

    Christian Goosmann & Britta Laube

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Contributions

Ö.P. cloned constructs and purified protomer for every experiment, crystallized, collected, processed and refined X-ray diffraction data and performed cellular assays; H.S. collected, processed and analyzed liquid-state NMR data and performed FTIR experiments and ThioflavinT binding assays; F.D. performed and analyzed DLS and X-ray fiber diffraction experiments; K.S. and C.A. collected, processed and analyzed solid-state NMR data; H.T. and Ö.P. purified and performed in vitro growth experiments with TTSS; C. Goosmann performed TEM studies; A.L. and M.B. designed and analyzed solid-state NMR experiments; C. Griesinger designed and analyzed liquid-state NMR experiments; A.T. designed and analyzed DLS and X-ray fiber diffraction experiments; V.B. designed TEM experiments; A.Z. designed functional and structural experiments; M.K. conceived this study, designed functional and TEM experiments, collected, refined and analyzed X-ray diffraction data and wrote the paper; all authors discussed the results and commented on the manuscript.

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Correspondence to Adam Lange or Michael Kolbe.

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Supplementary Figures 1–14, Supplementary Table 1 and Supplementary Methods (PDF 4245 kb)

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Poyraz, Ö., Schmidt, H., Seidel, K. et al. Protein refolding is required for assembly of the type three secretion needle. Nat Struct Mol Biol 17, 788–792 (2010). https://doi.org/10.1038/nsmb.1822

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