These authors contributed equally to this work. Final version of this article is published by NRC Research Press and available at: http://dx.doi.org/10.1139/ O2012-029 SummaryThe bacterial toxin pyolysin (PLO) belongs to the family of Cholesterol-Dependent Cytolysins (CDCs), which form large, oligomeric pores in cholesterol-containing membranes. Monomeric CDC molecules have a structure of four domains, with domains 2 and 3 packed against each other. After binding to target membranes containing cholesterol, toxin monomers oligomerize into pre-pore complexes. Trans-membrane pores form when the pre-pores insert into the lipid bilayer. Membrane insertion requires each subunit in the pre-pore to undergo a significant change in conformation, including the separation of domains 2 and 3. We here describe a pyolysin mutant with an engineered disulfide bond between domains 2 and 3 that is unable to complete this conformational change and thus does not form pores. In contrast to a similar mutant previously described for the homologous toxin perfringolysin O, the disulfide-tethered mutant does not form pre-pore oligomers, indicating that it is already inhibited at the stage of oligomerization. When mixed with wild type PLO, the mutant partially inhibits oligomerization of the latter, so that the resulting hybrid oligomers are reduced in size and arc-shaped rather than ring-shaped. Osmotic protection experiments indicate that such oligomers can form functional pores of reduced size. These findings indicate that conformational flexibility between domains 2 and 3 is required for oligomerization. Moreover, membrane insertion can be triggered in oligomers that contain some insertion-deficient subunits and is therefore only partially cooperative.2

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