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The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Nature Structural Biology volume 9, pages 476–484 (2002)Cite this article

Abstract

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.

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Figure 1: Planar lipid bilayer experiments with colicin E9.
Figure 2: Characteristics of E9 DNase channels.
Figure 3: E9 DNase channels at higher protein concentration.
Figure 4: Engineering a disulfide bond into the E9 DNase.
Figure 5: Effect of disulfide bond on E9 DNase channel activity.
Figure 6: 1H-15N HSQC spectra of E9 DNaseS–S and E9 DNaseSH2.
Figure 7: Antibacterial activity of oxidized and reduced colicin E9.

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Acknowledgements

We thank A. Reilly, C. Moore and N. Cull for expert technical assistance and A. Leech for help with the acquisition of all mass spectrometry data. We also thank the referees of this paper for their helpful comments. This work was supported by The Biotechnology and Biological Sciences Research Council. A.H.K. was supported by a Wellcome Trust Prize Studentship.

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Author notes

  1. Christelle Lemaître

    Present address: Laboratoire de Spectrométrie de Masse Bioorganique, Université Louis Pasteur, UMR/ULP CNRS 7509, ECPM 25 rue Becquerel, F-67087, Cedex 2, France

  2. Hamid Mobasheri

    Present address: Laboratory of Membrane Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384, IR, Iran

  3. Khédidja Mosbahi and Christelle Lemaître: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Khédidja Mosbahi, Christelle Lemaître, Anthony H. Keeble, Hamid Mobasheri, Edward J.A. Lea & Colin Kleanthous

  2. School of Chemical Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Bertrand Morel & Geoffrey R. Moore

  3. Division of Microbiology and Infectious Diseases, University Hospital, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK

    Richard James

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Mosbahi, K., Lemaître, C., Keeble, A. et al. The cytotoxic domain of colicin E9 is a channel-forming endonuclease. Nat Struct Mol Biol 9, 476–484 (2002). https://doi.org/10.1038/nsb797

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