title: Staphylococcal alpha-toxin: formation of the heptameric pore is partially cooperative and proceeds through multiple intermediate stages.
abstract: Staphylococcal alpha-toxin is a 293 residue polypeptide that assembles into pore-forming heptamers, residues 118-140, thereby inserting to form an amphipathic beta-barrel in the lipid bilayer. Fluorometric analyses were here conducted using cysteine-substitution mutants site-specifically-labeled at positions 35 or 130 with the environmentally-sensitive fluorophore acrylodan. In conjunction with functional assays, three conformational states of the heptamer were defined, which may represent transitional configurations of the toxin molecule along its way to membrane insertion and pore formation. The first was the freshly assembled, SDS-sensitive heptamer alpha7*a, where a minor alteration in the environment of H35 with no change in the environment of the membrane-inserting stem domain was observed. In transition stage alpha7*b, the stem domain moved from a hydrophilic to a more hydrophobic environment, due to protein-protein interaction. Transition to alpha7*c involved a cooperative effect, in which residue 35 was forced by a neighboring molecule into a markedly hydrophobic environment. At this stage, the heptamers acquired SDS stability. The final pore conformation alpha7 resulted when the stem domain inserted into the lipid bilayer, an event that was driven by H35 within the respective protomer. A model thus evolved in which cooperative forces first lever H35 into a position that subsequently drives the pore-forming sequence within each respective protomer into the membrane. In accord with this model, when hybrid heptamers were formed between a functionally defective H35 substitution mutant and active toxin, only the latter inserted their pore-forming domain into the membrane. In a satisfying functional correlate, pores of reduced size were then generated.
authors.affiliation: Institute of Medical Microbiology, University of Mainz, Hochhaus am Augustusplatz, D-55101 Mainz, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Valeva
authors.forename: M
authors.initials: M
authors.lastname: Palmer
authors.forename: S
authors.initials: S
authors.lastname: Bhakdi
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000595:Amino Acid Sequence / Q000235:genetics / Q000502:physiology; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000382:microbiology; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000737:chemistry / Q000235:genetics / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin; C038518:acrylodan; D015081:2-Naphthylamine