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. 1996 Apr 15;15(8):1857–1864.

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

A Valeva 1, A Weisser 1, B Walker 1, M Kehoe 1, H Bayley 1, S Bhakdi 1, M Palmer 1
PMCID: PMC450103  PMID: 8617232

Abstract

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arbuthnott J. P., Freer J. H., Billcliffe B. Lipid-induced polymerization of staphylococcal -toxin. J Gen Microbiol. 1973 Apr;75(2):309–319. doi: 10.1099/00221287-75-2-309. [DOI] [PubMed] [Google Scholar]
  2. Bayley H. Triggers and switches in a self-assembling pore-forming protein. J Cell Biochem. 1994 Oct;56(2):177–182. doi: 10.1002/jcb.240560210. [DOI] [PubMed] [Google Scholar]
  3. Bhakdi S., Füssle R., Tranum-Jensen J. Staphylococcal alpha-toxin: oligomerization of hydrophilic monomers to form amphiphilic hexamers induced through contact with deoxycholate detergent micelles. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5475–5479. doi: 10.1073/pnas.78.9.5475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bhakdi S., Muhly M., Füssle R. Correlation between toxin binding and hemolytic activity in membrane damage by staphylococcal alpha-toxin. Infect Immun. 1984 Nov;46(2):318–323. doi: 10.1128/iai.46.2.318-323.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhakdi S., Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991 Dec;55(4):733–751. doi: 10.1128/mr.55.4.733-751.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
  7. Fairweather N., Kennedy S., Foster T. J., Kehoe M., Dougan G. Expression of a cloned Staphylococcus aureus alpha-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus. Infect Immun. 1983 Sep;41(3):1112–1117. doi: 10.1128/iai.41.3.1112-1117.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Füssle R., Bhakdi S., Sziegoleit A., Tranum-Jensen J., Kranz T., Wellensiek H. J. On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. J Cell Biol. 1981 Oct;91(1):83–94. doi: 10.1083/jcb.91.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  10. Gouaux J. E., Braha O., Hobaugh M. R., Song L., Cheley S., Shustak C., Bayley H. Subunit stoichiometry of staphylococcal alpha-hemolysin in crystals and on membranes: a heptameric transmembrane pore. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12828–12831. doi: 10.1073/pnas.91.26.12828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gray G. S., Kehoe M. Primary sequence of the alpha-toxin gene from Staphylococcus aureus wood 46. Infect Immun. 1984 Nov;46(2):615–618. doi: 10.1128/iai.46.2.615-618.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  13. Jursch R., Hildebrand A., Hobom G., Tranum-Jensen J., Ward R., Kehoe M., Bhakdi S. Histidine residues near the N terminus of staphylococcal alpha-toxin as reporters of regions that are critical for oligomerization and pore formation. Infect Immun. 1994 Jun;62(6):2249–2256. doi: 10.1128/iai.62.6.2249-2256.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Krishnasastry M., Walker B., Braha O., Bayley H. Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal alpha-hemolysin. FEBS Lett. 1994 Dec 12;356(1):66–71. doi: 10.1016/0014-5793(94)01240-7. [DOI] [PubMed] [Google Scholar]
  15. Lakey J. H., Baty D., Pattus F. Fluorescence energy transfer distance measurements using site-directed single cysteine mutants. The membrane insertion of colicin A. J Mol Biol. 1991 Apr 5;218(3):639–653. doi: 10.1016/0022-2836(91)90707-d. [DOI] [PubMed] [Google Scholar]
  16. Palmer M., Jursch R., Weller U., Valeva A., Hilgert K., Kehoe M., Bhakdi S. Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. J Biol Chem. 1993 Jun 5;268(16):11959–11962. [PubMed] [Google Scholar]
  17. Palmer M., Weller U., Messner M., Bhakdi S. Altered pore-forming properties of proteolytically nicked staphylococcal alpha-toxin. J Biol Chem. 1993 Jun 5;268(16):11963–11967. [PubMed] [Google Scholar]
  18. Panchal R. G., Bayley H. Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis. J Biol Chem. 1995 Sep 29;270(39):23072–23076. doi: 10.1074/jbc.270.39.23072. [DOI] [PubMed] [Google Scholar]
  19. Prendergast F. G., Meyer M., Carlson G. L., Iida S., Potter J. D. Synthesis, spectral properties, and use of 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan). A thiol-selective, polarity-sensitive fluorescent probe. J Biol Chem. 1983 Jun 25;258(12):7541–7544. [PubMed] [Google Scholar]
  20. Shin Y. K., Levinthal C., Levinthal F., Hubbell W. L. Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants. Science. 1993 Feb 12;259(5097):960–963. doi: 10.1126/science.8382373. [DOI] [PubMed] [Google Scholar]
  21. Tobkes N., Wallace B. A., Bayley H. Secondary structure and assembly mechanism of an oligomeric channel protein. Biochemistry. 1985 Apr 9;24(8):1915–1920. doi: 10.1021/bi00329a017. [DOI] [PubMed] [Google Scholar]
  22. Valeva A., Palmer M., Hilgert K., Kehoe M., Bhakdi S. Correct oligomerization is a prerequisite for insertion of the central molecular domain of staphylococcal alpha-toxin into the lipid bilayer. Biochim Biophys Acta. 1995 Jun 14;1236(2):213–218. doi: 10.1016/0005-2736(95)00060-g. [DOI] [PubMed] [Google Scholar]
  23. Walker B., Bayley H. Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J Biol Chem. 1995 Sep 29;270(39):23065–23071. doi: 10.1074/jbc.270.39.23065. [DOI] [PubMed] [Google Scholar]
  24. Walker B., Kasianowicz J., Krishnasastry M., Bayley H. A pore-forming protein with a metal-actuated switch. Protein Eng. 1994 May;7(5):655–662. doi: 10.1093/protein/7.5.655. [DOI] [PubMed] [Google Scholar]
  25. Walker B., Krishnasastry M., Bayley H. Functional complementation of staphylococcal alpha-hemolysin fragments. Overlaps, nicks, and gaps in the glycine-rich loop. J Biol Chem. 1993 Mar 5;268(7):5285–5292. [PubMed] [Google Scholar]
  26. Walker B., Krishnasastry M., Zorn L., Bayley H. Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis. J Biol Chem. 1992 Oct 25;267(30):21782–21786. [PubMed] [Google Scholar]
  27. Ward R. J., Palmer M., Leonard K., Bhakdi S. Identification of a putative membrane-inserted segment in the alpha-toxin of Staphylococcus aureus. Biochemistry. 1994 Jun 14;33(23):7477–7484. doi: 10.1021/bi00189a056. [DOI] [PubMed] [Google Scholar]

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