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Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit β-strand alignment

Nature Structural & Molecular Biology volume 11, pages 697705 (2004) | Download Citation



Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores. Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques. Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden β-strand that forms the monomer-monomer interface and is required for oligomer assembly. The β-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane β-hairpins of adjacent monomers in the proper register for insertion. Collectively, these data reveal a novel membrane binding–dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.

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  1. 1.

    Introduction to the family of the structurally related cholesterol-binding cytolysins ('sulfhydryl-activated' toxins). In The Comprehensive Sourcebook of Bacterial Protein Toxins (eds. Alouf, J.E. & Freer, J.H.) 443–456 (Academic, London, 1999).

  2. 2.

    , & The cholesterol-dependent cytolysins. Curr. Top. Microbiol. Immunol. 257, 15–33 (2001).

  3. 3.

    , & The projection structure of perfringolysin O (Clostridium perfringens Θ-toxin). FEBS Lett. 319, 125–127 (1993).

  4. 4.

    , , , & Structure of a cholesterol-binding, thiol-activated cytolysin and a model of its membrane form. Cell 89, 685–692 (1997).

  5. 5.

    , & β-barrel pore forming toxins: intriguing dimorphic proteins. Biochemistry 40, 9065–9073 (2001).

  6. 6.

    & Pore-forming protein structure analysis in membranes using MIFT, multiple independent fluorescence techniques. Cell Biochem. Biophys. 36, 89–102 (2002).

  7. 7.

    et al. Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an α-helical to β-sheet transition identified by fluorescence spectroscopy. Biochemistry 37, 14563–14574 (1998).

  8. 8.

    et al. The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins. Cell 99, 293–299 (1999).

  9. 9.

    , , & Mechanism of membrane insertion of a multimeric β-barrel protein: Perfringolysin O creates a pore using ordered and coupled conformational changes. Mol. Cell 6, 1233–1242 (2000).

  10. 10.

    , , & Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin. Nat. Struct. Biol. 9, 823–827 (2002).

  11. 11.

    , , & The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane β-hairpins. Biochemistry 39, 10284–10293 (2000).

  12. 12.

    et al. Arresting pore formation of a cholesterol-dependent cytolysin by disulfide trapping synchronizes the insertion of the transmembrane β-sheet from a prepore intermediate. J. Biol. Chem. 276, 8261–8268 (2001).

  13. 13.

    et al. Monomer-monomer interactions drive the prepore to pore conversion of a β -barrel-forming cholesterol-dependent cytolysin. J. Biol. Chem. 277, 11597–11605 (2002).

  14. 14.

    , & Assembly and topography of the prepore complex in cholesterol-dependent cytolysins. J. Biol. Chem. 278, 31218–31225 (2003).

  15. 15.

    , & The signal sequence moves through a ribosomal tunnel into a noncytoplasmic aqueous environment at the ER membrane early in translocation. Cell 73, 1101–1115 (1993).

  16. 16.

    Intramolecular pyrene excimer fluorescence: a probe of proximity and protein conformational change. Methods Enzymol. 278, 286–295 (1997).

  17. 17.

    A possible role for π-stacking in the self-assembly of amyloid fibrils. FASEB J. 16, 77–83 (2002).

  18. 18.

    & Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation. Proc. Natl. Acad. Sci. USA 99, 2754–2759 (2002).

  19. 19.

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

  20. 20.

    & Raster3D photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

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This work was supported by US National Institutes of Health grant AI 37657 and the Robert A. Welch Foundation.

Author information


  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.

    • Rajesh Ramachandran
    •  & Arthur E Johnson
  2. Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.

    • Rodney K Tweten
  3. Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.

    • Arthur E Johnson
  4. Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.

    • Arthur E Johnson


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The authors declare no competing financial interests.

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Correspondence to Arthur E Johnson.

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