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Mosbahi, Khédidja; Lemaître, Christelle; Mobasheri, Hamid; Morel, Bertrand; Lea, Edward J.A.; Kleanthous, Colin; Keeble, Anthony H.; James, Richard; Moore, Geoffrey R. (2002)
Languages: English
Types: Article
Subjects:
Bacterial toxins commonly translocate cytotoxic enzymes into cells using dedicated channelforming subunits or domains as conduits. We demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (the E9 DNase) exhibits nonvoltage- gated, channel-forming activity in planar lipid bilayers and that this activity 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, with NMR experiments suggesting decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond was 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.
  • The results below are discovered through our pilot algorithms. Let us know how we are doing!

    • 1School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K 2Present Address: Laboratoire de Spectrométrie de Masse Bioorganique, Université Louis Pasteur, UMR/ULP CNRS 7509, ECPM 25 rue Becquerel, F-67087 Cedex 2, France.
    • 3Present Address: Laboratory of Membrane Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, PO Box 13145-1384, IR Iran.
    • 4School of Chemical Sciences, University of East Anglia, Norwich, NR4 7TJ, U.K 5Division of Microbiology and Infectious Diseases, University Hospital, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, U.K.
    • 6These authors contributed equally to this work.
    • 12. Pommer, A.J., Wallis, R., Moore, R., James, R. & Kleanthous, C. Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9. Biochem. J. 334, 387-392 (1998).
    • 13. Oh, J.K., Senzel, L., Collier, R.J. & Finkelstein, A. Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain. Proc. Natl. Acad. Sci. USA 96, 8467-8470 (1999).
    • 14. Wallis, R. et al. In vivo and in vitro characterization of overproduced colicin E9 immunity protein. Eur. J. Biochem. 207, 687-695 (1992).
    • 15. Kleanthous, C. & Walker, D. Immunity Proteins: Enzyme inhibitors that avoid the active site. Trends Biochem. Sci. 26, 624-631 (2001).
    • 16. Penfold, C.N. et al. A 76 residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent E.coli 113/3 cells. Mol. Microbiol. 38, 639-649 (2000).
    • 17. Wallis, R. et al. Tandem overexpression and characterization of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9. Eur. J. Biochem. 220, 447-454 (1994).
    • 18. Slatin, S.L., Nardi, A., Jakes, K., Baty, D. & Duché, D. Translocation of a functional protein by a voltage-dependent ion channel. Proc. Natl. Acad. Sci. USA 99, 1286-1291 (2002).
    • 19. Jakes, K.S., Kienker, P.K. & Finkelstein, A. Channel-forming colicins: translocation (and other deviant behaviour) associated with colicin Ia channel gating. Quat. Rev. Biophys. 32, 189-205 (1999).
    • 20. Raymond, L., Slatin, S.L. & Finkelstein, A. Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent selectivity. J. Membr. Biol. 84, 173- 181 (1985).
    • Bullock, J.O. & Kolen, E.R. Ion selectivity of colicin E1. J. Membr. Biol. 144, 131-145 (1995).
    • Kadner, R.J. In Escherichia coli and Salmonella; cellular and molecular biology 2nd Edition, (ed. Neidhardt, F.C.), Vol. 1, 58-87 (ASM Press, Washington; 1996).
    • Kleanthous, C. et al. Structural and mechanistic basis of immunity towards endonuclease colicins. Nature Struct. Biol. 6, 243-252 (1999).
    • Pommer, A.J. et al. Homing-in on the role of transition metals in the HNH motif of colicin endonucleases. J. Biol. Chem. 274, 27153-27160 (1999).
    • Wallis, R., Moore, G.R., James, R. & Kleanthous, C. Protein-protein interactions in colicin E9 DNase-immunity protein complexes. Diffusion-controlled association and femtomolar binding for the cognate complex. Biochemistry 34, 13743-13750 (1995).
    • Pommer, A.J. et al. Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9. J. Mol. Biol. 314, 735-749 (2001).
    • Biol. Chem. 269, 6332-6339 (1994).
    • Kühlmann, U.C., Pommer, A.J., Moore, G.R., James, R., & Kleanthous, C. Specificity in protein-protein interactions: The structural basis for dual recognition in colicin endonuclease-immunity protein complexes. J. Mol. Biol. 301, 1163-1178 (2000).
    • Whittaker, S.B.M. et al. NMR detection of slow conformational dynamics in an endonuclease toxin. J. Biomol. NMR 12, 145-159 (1998).
    • 31. Wallis, R. et al. Protein-protein interactions in colicin E9 DNase-immunity protein complexes. Cognate and noncognate interactions that span the mM-fM affinity range. Biochemistry 34, 13751-13759 (1995).
    • 32. Garinot-Schneider, C., Pommer, A.J., Moore, G.R., Kleanthous, C. & James, R. Identification of putative active-site residues in the DNase domain of colicin E9 by random mutagenesis. J. Mol. Biol. 260, 731-742 (1996).
    • 33. Lacy, B.D. & Stevens, R.C. Unraveling the structures and modes of action of bacterial toxins. Curr. Op. Struct. Biol. 8, 778-784 (1998).
    • 34. Falnes, P.Ø. & Sandvig, K. Penetration of protein toxins into cells. Curr. Op. Cell Biol. 12, 407-413 (2000).
    • 35. Chevalier, B.S. & Stoddard, B.L. Homing endonucleases: structural and functional insight into the catalysts of intron/intein mobility. Nucl. Acid. Res. 29, 3757-3774 (2001).
    • 36. van der Goot, F.G, González-Mañas, J.M., Lakey, J.H. & Pattus, F. A ‘molten-globule’ membrane insertion intermediate of the pore-forming domain of colicin A. Nature 354, 408-410 (1991).
    • 37. Zakharov, S.D. et al. Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc. Natl. Acad. Sci. USA 95, 4282-4287 (1998).
    • 38. Slatin, S.L., Qiu, X-Q., Jakes, K.S., & Finkelstein, A. Identification of a translocated protein segment in a voltage-dependent channel. Nature 371, 158-161 (1998).
    • 39. de Zamaroczy, M., Mora, L., Lecuyer, A., Géli, V. & Buckingham, R.H. Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Mol. Cell 8, 159-168 (2001).
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