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Structure of the key toxin in gas gangrene

Nature Structural Biology volume 5, pages 738–746 (1998)Cite this article

Abstract

Clostridium perfringens α-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with α-toxin. Finally, a possible mechanism for α-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.

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Figure 1: a, Cartoon representation of the α-toxin chain.
Figure 2: Sequence alignment of α-toxin with homologous or structurally similar proteins.
Figure 3
Figure 4: Stereo view of the active site, showing metal ions as grey spheres.
Figure 5: GRASP40 electrostatic potential surface of α-toxin (shown with the active site cleft and membrane-binding surface uppermost).
Figure 6: Model for membrane binding.
Figure 7: Comparison of α-toxin C-terminal domain with other similar folds.
Figure 8: Stereo view of 2Fo - Fc omit map for the metal ions in the catalytic domain, together with the final co-ordinates.

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References

  1. MacFarlane, M.G. & Knight, B.C.J.G. The biochemistry of bacterial toxins. I. Lecinthinase activity of CI. welchii toxins. Biochem. J. 35, 884– 902 (1941).

    Article  CAS  Google Scholar 

  2. Hough, E. et al. High resolutions (1.5 Å) crystal structure of phospholipase C from Bacillus cereus. Nature 338, 357– 360 (1989).

    Article  CAS  Google Scholar 

  3. Williamson, E.D. & Titball, R.W. A genetically engineered vaccine against the alpha-toxin of Clostridium perfringens also protects mice against experimental gas gangrene. Vaccine 11, 1253– 1258 (1993).

    Article  CAS  Google Scholar 

  4. Awad, M.M., Bryant, A.E., Stevens, D.L. & Rood, J.I. Virulence studies on chromosomal α-toxin and α-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α-toxin in Clostridium perfringens-mediated gas gangrene. Mol. Microbiol . 15, 191– 202 ( 1995).

    Article  CAS  Google Scholar 

  5. Titball, R.W. Bacterial phospholipases C. Microbiol. Rev. 57, 347 – 366 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Titball, R.W., Fearn, A.M. & Williamson, E.D. Biochemical and immunological properties of the C-terminal domain of the alpha-toxin of Clostridium perfringens. FEMS Microbiol Lett. 110, 45– 50 (1993).

    Article  CAS  Google Scholar 

  7. Nalefski, E.A. & Falke, J.J. The C2 domain calcium binding motif: structural and functional diversity. Prot. Sci. 12, 2375– 2390 (1996).

    Article  Google Scholar 

  8. Basak, A.K. et al. Crystallisation and preliminary X-ray diffraction studies of α-toxin from two different strains (NCTC-8237 and CER89L43) of Clostridium perfringens . Acta Crystallogr. in the press ( 1998).

  9. Brünger, A.T. X-PLOR Manual, Version 3.1. A system for X-ray crystallography and NMR (Yale University Press, New Haven, Connecticut; 1992).

    Google Scholar 

  10. Nagahama, M., Okagawa, Y., Nakayama, T., Nishioka, E. & Sakurai, J. Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin. J. Bacteriol. 177, 1179– 1183 (1995).

    Article  CAS  Google Scholar 

  11. Weast, R.C. (ed.) CRC Handbook of Chemistry and Physics, 49th Edition (The Chemical Rubber Company, Cleveland, Ohio; 1969).

    Google Scholar 

  12. Young, P.R., Snyder, W.R. and McMahon, R. Kinetic mechanism of Clostridium perfringens phospholipase C. Biochem. J. 280, 407– 410 (1992).

    Article  Google Scholar 

  13. Byberg, J.R., Jorgensen, F.S., Hansen, S. & Hough, E. Substrate-enzyme interactions and catalytic mechanism in phospholipase C: a molecular modelling study using the GRID program. Proteins 12, 331– 338 (1992).

    Article  CAS  Google Scholar 

  14. Goodford, P.J. A computational procedure for determining energetically favorable binding sites on biologically important molecules. J. Med. Chem. 28, 849 – 857 (1985).

    Article  CAS  Google Scholar 

  15. Nagahama, M., Michiue, K. & Sakurai, J. Membrane-damaging action of Clostridium perfringens alpha-toxin on phospholipid liposomes. Biochim. Biophys. Acta. 1280 , 120– 126 (1996).

    Article  Google Scholar 

  16. Guillouard, I., Alzari, P.M., Sailou, B. and Cole, S.T. The carboxy-terminal C2-like domain of the α-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition. Mol. Microbiol. 26 867– 876 ( 1997).

    Article  CAS  Google Scholar 

  17. Winkler, F.K., D'Arcy, A. & Hunziker, W. Structure of human pancreatic lipase. Nature 343, 771– 774 (1990).

    Article  CAS  Google Scholar 

  18. Minor, W. et al. Crystal structure of soybean lipoxygenase-1 at 1.4 Å resolution. Biochemistry 35 10687– 10701 (1996).

    Article  CAS  Google Scholar 

  19. Sutton, R.B. Davletox, B.A., Berghuis, A.M., Südhof, T.C. & Sprang, S.R. Structure of the first C2 domain of Synaptotagmin I: A novel Ca2+/phospholipid binding fold. Cell 80, 929– 938 ( 1995).

    Article  CAS  Google Scholar 

  20. Essen, L.-O., Perisic, O., Cheung, R., Katan, M. & Williams, R.-L. Crystal structure of a mammalian phosphoinositide-specific phospholipase Cγ1. Nature 380, 595 – 602 (1996).

    Article  CAS  Google Scholar 

  21. Essen, L.-O., Persic, O., Lynch, D.E., Katan, M. and Williams, R.L. A ternary metal binding site in the C2 domain of phosphoinositide-specific phospholipase C-γ1. Biochemistry 36 2753– 2762 (1997).

    Article  CAS  Google Scholar 

  22. Shau. XI, Davletov, B.A., Sutton, R.B., Südhof, T.C. and Rizo, J. Bipartite Ca2+-binding motif in C2 domains of Synaptotagmin and Protein Kinase C. Science 273 248– 251 ( 1996).

    Article  Google Scholar 

  23. Gilmor, S.A., Villaseñor, A., Fletterick, R., Sigal, E. and Browner, M.F. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. NSB 4 1003– 1009 ( 1997).

    Google Scholar 

  24. van Tilbeurgh, H., Sarda, L., Verger, R. & C. Cambillau, C. Structure of the pancreatic lipase-procolipase complex. Nature 359, 159 (1992).

    Article  CAS  Google Scholar 

  25. Naguchi, M., Miyano, M., Natsumoto, T. and Nama, M. Human 5-lipoxygenase associates with phosphatidylcholine liposomes and modulates LTA4 synthetase activity. Biochim. Biophys. Acta 1215 300 – 306 (1994).

    Article  Google Scholar 

  26. Perisic, O., Fong, S., Lynch, D.E., Bycroft, M. and Williams, R.L. Crystal structure of a calcium-phospholipid binding domain from cytsolic phospholipase A2. J. Biol. Chem. 273 1596– 1604 (1998).

    Article  CAS  Google Scholar 

  27. Otnæss, A.-B . et al. Some characteristics of phospholipase C from Bacillus cereus . Eur. J. Biochem. 79, 459– 468 (1977).

    Article  Google Scholar 

  28. Otwinowski, Z. Proceedings of the CCP4 study weekend: Data Collection and Processing (eds L. Sawyer, N. Isaacs, S. Bailey) 56– 62 (SERC Daresbury Laboratory, Warrington, UK; 1993).

    Google Scholar 

  29. Collaborative Computational Project, No 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760– 763 (1994).

  30. Navaza, J. AMoRe: an automated packate for molecular replacement. Acta Crystallogr. A50, 157– 163 (1994).

    Article  CAS  Google Scholar 

  31. Cowtan, K. 'dm': An automated procedure for phase improvement by density modification. Joint CCP4 and ESRF-EACBM Newsletter on Protein Crystallography 34, 946– 950 (1991).

    Google Scholar 

  32. Esnouf, R. An extensively modified version of Molscript that includes greatly modified colouring capabilities . J. Mol. Graphics 15, 132– 134 (1997).

    Article  CAS  Google Scholar 

  33. Kraulis, P. Molscript: A program to produce both detailed and schematic plots of protein structures . J. Appl. Crystallogr. 24, 946– 950 (1991).

    Article  Google Scholar 

  34. Merrit, E.A. & Murphy, M.E.P. Raster3D Version 2.0 - A program for photorealistic molecular graphics. Acta Crystallogr. D50, 869– 873 (1994).

    Google Scholar 

  35. Bacon, D.J. & Anderson, W.F. A fast algorithm for rendering space-filling molecule pictures. J. Mol. Graph. 6, 219– 220 (1988).

    Article  Google Scholar 

  36. Tso, J.Y. & Siebel, C. Cloning and expression of the phospholipase C gene from Clostricium perfringens and Clostricium bifermentans . Infect. Immun. 57, 468– 476 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Glimore, M.S., Cruz-Rodz, A.L., Leimeister-Wachter, M., Kreft, J. & Goebel, W. A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes; nucleotide sequence and genetic linkage. J. Bacteriol. 171, 744– 753 (1989).

    Article  Google Scholar 

  38. Matsumoto, T. et al. Molecular cloning and amino acid sequence of human arachidonate 5-lipoxygenase . Adv. Prostoglandin Thromboxane Leukot. Res. 19,– 466 (1989).

  39. Suh, P.G., Ryu, S.H., Moon, K.H., Suh, H.W. and Rhee S.G. Cloning and sequence of multiple forms of phosphlipase C. Cell 54 161– 169 ( 1988).

    Article  CAS  Google Scholar 

  40. Nicholls, A., Sharp, K. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281– 296 (1991).

    Article  CAS  Google Scholar 

  41. Read, R.J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A42, 140– 149 (1986).

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank D. I. Stuart (Laboratory of Molecular Biophysics, Oxford University) for help with the original structure and J. Thornton (University and Birkbeck Colleges, London University) for useful comments on the manuscript, R. Esnouf (Rega Institute for Medical Research, Catholic University of Leuven, Belgium) for the most recent version of Bobscript and also G. Wright for help with data collection. This research is supported by a grant from the BBSRC, and computers used in the structure solution were provided by the Wellcome Trust.

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Authors and Affiliations

  1. Department of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, England

    Claire E. Naylor, Julian T. Eaton, Neil Justin, David S. Moss & Ajit K. Basak

  2. Defence Evaluation and Research Agency, CBD Porton Down, Salisbury, SP4 0QJ, Wiltshire, England

    Angela Howells & Richard W. Titball

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  1. Claire E. Naylor
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Correspondence to Ajit K. Basak.

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Naylor, C., Eaton, J., Howells, A. et al. Structure of the key toxin in gas gangrene. Nat Struct Mol Biol 5, 738–746 (1998). https://doi.org/10.1038/1447

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