title: Solubilization and characterization of the anthrax toxin pore in detergent micelles.
abstract: Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self-associates to form a heptameric ring-shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore-like state. N-tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion-competent 440-kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore-like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14-solubilized pore showed an extended, mushroom-shaped structure. Circular dichroïsm measurements revealed an increase in alpha helix and a decrease in beta structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2beta2-2beta3 loop transforms into the transmembrane segment of the beta-barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to alpha structure is dependent on residues of the luminal 2beta11-2beta12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Ave., Boston, Massachusetts 02115, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Vernier
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Laura D
authors.initials: LD
authors.lastname: Jennings
authors.forename: Jianjun
authors.initials: J
authors.lastname: Sun
authors.forename: Audrey
authors.initials: A
authors.lastname: Fischer
authors.forename: Likai
authors.initials: L
authors.lastname: Song
authors.forename: R John
authors.initials: RJ
authors.lastname: Collier
mesh_terms: D000942:Antigens, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D001408:Bacillus anthracis / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D011487:Protein Conformation; D012995:Solubility
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; C030325:anthrax toxin