The mechanism of membrane insertion for a cholesterol-dependent cytolysin:A novel paradigm for pore-forming toxins

Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pat hogenic Clostridium perfringens, oligomerizes and forms large pores upon en countering cholesterol-containing membranes. Whereas all pore-forming bacte rial toxins examined previously have been shown to penetrate the membrane u sing a single amphipathic beta hairpin per polypeptide, cysteine-scanning m utagenesis and multiple independent fluorescence techniques here reveal tha t each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic a hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six a lpha helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a nove l paradigm far the mechanism of membrane insertion by a cytolytic toxin.