Article | Published:

Membrane insertion of a Tc toxin in near-atomic detail

Nature Structural & Molecular Biology volume 23, pages 884890 (2016) | Download Citation

Abstract

Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

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Acknowledgements

We thank K. Vogel-Bachmayr for assistance with site-directed mutagenesis and cloning, and A. Elsner for technical support. We thank O. Hofnagel for excellent assistance in electron microscopy. We gratefully acknowledge R. Matadeen and S. de Carlo (FEI Company) for image acquisition at the Netherlands Centre for Nanoscopy in Leiden (NeCEN), which is cofinanced by grants from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (project 175.010.2009.001) and by the European Union's Regional Development Fund through 'Kansen voor West' (project 21Z.014). This work was supported by funds from the Max Planck Society (to S.R.) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013) (grant no. 615984) (to S.R.). We thank R. Shaw (Cardiovascular Research Institute, University of California San Francisco) for reagents.

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Affiliations

  1. Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany.

    • Christos Gatsogiannis
    • , Felipe Merino
    • , Daniel Prumbaum
    • , Daniel Roderer
    • , Franziska Leidreiter
    • , Dominic Meusch
    •  & Stefan Raunser

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Contributions

S.R. designed the project. D.M. designed and purified proteins. C.G. and F.L. optimized the protein-nanodisc preparation for data collection. C.G. performed sample preparation, collected negative-stain data, processed and refined cryo-EM data, built the atomic model and analyzed the data. F.M. calculated MD simulations and analyzed the data. D.P. and D.R. performed mutational studies and the liposome-based membrane activity assay. D.P. performed live-cell imaging. C.G. and F.M. prepared the figures. S.R. managed the project, analyzed data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Stefan Raunser.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Note

  2. 2.

    Supplementary Data Set 1

    Purification of wild type and mutant TcdA1 proteins.

Videos

  1. 1.

    Cryo-EM map of TcA in its pore state embedded in nanodiscs.

    The cryo-EM map of TcA from Photorhabdus luminescens, colored by subunits and filtered according to local resolution (see Supplementary Fig. 1 for details), is rotated to show the overall structure and zoomed in on representative regions, in particular an α-helix and a β-strand. Last, the molecular model of the linker (entropic spring) is shown superimposed with the respective cryoEM density.

  2. 2.

    Conformational change of the transmembrane domain from prepore to pore state.

    The video focuses on the conformational changes of the transmembrane domain showing the membrane-induced opening of the pore. The narrowest site of the prepore lumen (Tyr 2,163) is shown in sticks

  3. 3.

    Interaction between the transmembrane region of TcA and the lipid head groups.

    The animation includes 0.5 μs of simulation,where the lipid head groups recurrently intercalate between the helices of the protein. We highlighted the intercalated lipids for a representative frame located around the middle of the simulation.

  4. 4.

    Syringe-like injection and channel opening mechanism.

    The video shows a simplified model of the syringe-like injection, membrane penetration and channel opening, obtained after morphing between the structure of TcA in the prepore and the pore state. It should be noted, that a structure of a possible intermediate state (with the toxin inserted in the membrane but the channel in the prepore state), as shown in this animation, is not available yet.

  5. 5.

    Conformational change between prepore to pore TcA protomer.

    The video shows a morph between the structures a TcA protomer in the prepore and pore state. The conformational changes of the entropic spring are highlighted.

  6. 6.

    Representative trajectories along the linker extension free energy calculations.

    States near the pore, and prepore extensions, as well as an intermediate state are shown. The blue spheres show the atoms used to measure the end-to-end distance of the molecule. For guidance, the positions of the trajectories along the free energy profile are highlighted.

  7. 7.

    Membrane deformation during the coarse-grained MD simulations of membrane penetration.

    The trajectories for each of state shown in Fig. 5 are included. For guidance, the position of the windows along the free energy profile is indicated with an arrow.

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DOI

https://doi.org/10.1038/nsmb.3281

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