Article | Published:

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

Subjects

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  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).

Download references

Acknowledgements

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

Author information

Affiliations

  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

Authors

  1. Search for Rajesh Ramachandran in:

  2. Search for Rodney K Tweten in:

  3. Search for Arthur E Johnson in:

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Arthur E Johnson.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb793

Further reading