title: Partial oligomerization of pyolysin induced by a disulfide-tethered mutant.
abstract: The bacterial toxin pyolysin (PLO) belongs to the family of cholesterol-dependent cytolysins (CDCs), which form large, ring-shaped 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 characterize a pyolysin mutant with an engineered disulfide bond between domains 2 and 3. The disulfide-tethered mutant binds to membranes but does not form oligomers. When mixed with wild type PLO, the two proteins form hybrid oligomers, which are reduced in size and arc-shaped rather than ring-shaped. With equimolar mixtures or the disulfide mutant in slight excess, the hybrid oligomers retain pore-forming activity, while a larger excess of the mutant suppresses pore formation. These results support a "partially cooperative" mode of protein activity, in which a limited number of functional subunits within an oligomer have to cooperate to initiate membrane insertion and pore formation.
authors.affiliation: Department of Chemistry, University of Waterloo, 200 University Ave. W, Waterloo, ON N2L 3G1, Canada.
authors.forename: Lisa
authors.initials: L
authors.lastname: Pokrajac
authors.forename: Clara
authors.initials: C
authors.lastname: Baik
authors.forename: J Robin
authors.initials: JR
authors.lastname: Harris
authors.forename: Naghmeh S
authors.initials: NS
authors.lastname: Sarraf
authors.forename: Michael
authors.initials: M
authors.lastname: Palmer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D058105:Polymerization; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D004220:Disulfides; D006460:Hemolysin Proteins; D021122:Protein Subunits; C108448:Pyolysin, Arcanobacterium pyogenes