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Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Abstract

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes α-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

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References

  1. Cabiaux, V., Brasseur, R., Wattiez, R., Falmagne, P., Ruysschaert, J.M., Goormaghtigh, E. 1989. Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy. J. Biol. Chem. 264:4928–4938

  2. Cabiaux, V., Mindell, J.A., Collier, R.J. 1993. Involvement of Isoleucine 364 in the translocation of diphtheria toxin: relationship between cytotoxicity and pore formation. Infection and Immunity 61:2200–2202

  3. Choe, S., Bennett, M.J., Fujii, G., Curmi, P.M., Kantardjieff, K.A., Collier, R.J., Eisenberg, D. 1992. The crystal structure of diphtheria toxin. Nature 357:216–222

  4. Collier, R.J. 1990. Diphtheria toxin: Structure and function of a cytocidal protein. In: ADP-Ribosylating Toxins and G-Proteins: Insights into Signal Transduction. J. Moss and M. Vaughn, editors, pp. 3–19. Am. Soc. Microbiol., Washington, DC

  5. Deleers, M., Beugnier, N., Falmagne, P., Cabiaux, V., Ruysschaert, J.M. 1983. Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH. FEBS Lett. 160:82–86

  6. Draper, R.K., Simon, M.I. 1980. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J. Cell Biol. 87:849–854

  7. Falnes, P.O., Madshus, I.H., Sandvig, K., Olsnes, S. 1992. Replacement of negative by positive charges in the presumed membraneinserted part of diphtheria toxin B fragment. Effect on membrane translocation and on formation of cation channels. J. Biol. Chem. 267:12284–12290

  8. Hoch, D.H. 1985. Botulinum, Tetanus, and Diphtheria Toxin Channels in Planar Lipid Bilayers. Ph.D. Thesis. Albert Einstein College of Medicine, Yeshiva University, Bronx, NY

  9. Hoch, D.H., Finkelstein, A. 1985. Gating of large toxin channels by pH. Ann. NY Acad. Sci. 456:33–35

  10. Hoch, D.H., Romero-Mira, M., Ehrlich, B.E., Finkelstein, A., DasGupta, B.R., Simpson, L.L. 1985 Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes. Proc. Natl. Acad. Sci. USA 82:1692–1696

  11. Kagan, B.L., Finkelstein, A., Colombini, M. 1981. Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc. Natl. Acad. Sci. USA 78:4950–4954

  12. Kagawa, Y., Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation. XXV. Reconstitution of vesicles catalyzing 32Pi-adenosine triphosphate exchange. J. Biol. Chem. 246:5477–5487

  13. Kayser, G., Lambotte, P., Falmagne, P., Capiau, C., Zanen, J., Ruysschaert, J.M. 1981. A CNBR peptide located in the middle region of diphtheria toxin fragment B induces conductance change in lipid bilayers. Possible role of an amphipathic helical segment. Biochem. Biophys. Res. Commun. 99:358–363

  14. London, E. 1992. Diphtheria toxin: membrane interaction and membrane translocation. Biochim. Biophys. Acta 1113:25–51

  15. Madshus, I.H., Stenmark, H. 1992. Entry of ADP-ribosylating toxins into cells. Curr. Top. Microbiol. Immunol. 175:1–26

  16. Mindell, J.A., Silverman, J.A., Collier, R.J., Finkelstein, A. 1992. Locating a residue in the diphtheria toxin channel. Biophys. J. 62:41–44

  17. Mindell, J.A., Silverman, J.A., Collier, R.J., Finkelstein, A. 1994a. Structure-function relationships in the diphtheria toxin channel: II. A residue responsible for the channel's dependence on trans pH. J. Membrane Biol. 137:29–44

  18. Mindell, J.A., Silverman, J.A., Collier, R.J., Finkelstein, A. 1994b. Structure-function relationships in the diphtheria toxin channel: III. Residues which affect the cis pH dependence of channel conductance. J. Membrane Biol. 137:45–57

  19. Misler, S. 1983. Gating of ion channels made by a diphtheria toxin fragment in phospholipid bilayer membranes. Proc. Natl. Acad. Sci. USA 80:4320–4324

  20. Montal, M. 1974. Formation of bimolecular membranes from lipid monolayers. Methods Enzymol. 32:545–554

  21. O'Keefe, D.O., Cabiaux, V., Choe, S., Eisenberg, D., Collier, R.J. 1992. pH-dependent insertion of proteins into membranes: Bchain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349---Lys. Proc. Natl. Acad. Sci. USA 89:6202–6206

  22. Papini, E., Sandona, D., Rappuoli, R., Montecucco, C. 1988. On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells. EMBO J. 7:3353–3359

  23. Pappenheimer, A.M., Jr., Uchida, T., Harper, A.A. 1972. An immunological study of the diphtheria toxin molecule. Immunochemistry 9:891–906

  24. Riggs, P. 1990. Expression and purification of maltose binding protein fusions. In: Current Protocols in Molecular Biology. Ausbel et al., editors. Wiley Interscience, Cambridge, MA

  25. Sandvig, K., Olsnes, S. 1980. Diphtheria toxin entry into cells is facilitated by low pH. J. Cell Biol. 87:828–832

  26. Sandvig, K., Olsnes, S. 1988. Diphtheria toxin-induced channels in Vero cells selective for monovalent cations. J. Biol. Chem. 263:12352–12359

  27. Tweten, R.K., Barbieri, J.T., Collier, R.J. 1985. Diphtheria toxin. Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltransferase activity. J. Biol. Chem. 260:10392–10394

  28. Uchida, T., Pappenheimer, A.M., Jr., Greany, R. 1973. Diphtheria toxin and related proteins. I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. J. Biol. Chem. 248:3838–3844

  29. Uchida, T., Pappenheimer, A.M., Jr., Harper, A.A. 1972. Reconstitution of diphtheria toxin from two nontoxic cross-reacting mutant proteins. Science 175:901–903

  30. vanderSpek, J.C., Mindell, J.A., Finkelstein, A., Murphy, J.R. 1993. Structure/function analysis of the transmembrane domain of DAB(389)-Il-2, an interleukin 2 receptor-targeted fusion toxin. J. Biol. Chem. 268:12077–12082

  31. Williams, D.P., Snider, C.E., Strom, T.B., Murphy, J.R. 1990. Structure/function analysis of interleukin-2-toxin (DAB486-IL-2). Fragment B sequences required for the delivery of fragment A to the cytosol of target cells. J. Biol. Chem. 265:11885–11889

  32. Wonderlin, W.F., Finkel, A., French, R.J. 1990. Optimizing planar lipid bilayer single-channel recordings for high time resolution. Biophys. J. 58:289–297

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Additional information

This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).

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Silverman, J.A., Mindell, J.A., Zhan, H. et al. Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain. J. Membarin Biol. 137, 17–28 (1994). https://doi.org/10.1007/BF00234995

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Key words

  • Diphtheria toxin
  • Site-directed mutagenesis
  • Planar lipid bilayers
  • Ion channels
  • T-domain
  • Channel-forming peptides