title: Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure.
abstract: Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.
authors.affiliation: Institute of Medical Microbiology and Hygiene, University of Mainz, Augustusplatz, D55101 Mainz, Germany. avaleva@mail.uni-mainz.de
authors.forename: Angela
authors.initials: A
authors.lastname: Valeva
authors.forename: Iwan
authors.initials: I
authors.lastname: Walev
authors.forename: Fatima
authors.initials: F
authors.lastname: Boukhallouk
authors.forename: Trudy M
authors.initials: TM
authors.lastname: Wassenaar
authors.forename: Natalia
authors.initials: N
authors.lastname: Heinz
authors.forename: Julia
authors.initials: J
authors.lastname: Hedderich
authors.forename: Sina
authors.initials: S
authors.lastname: Lautwein
authors.forename: Moritz
authors.initials: M
authors.lastname: Möcking
authors.forename: Silvia
authors.initials: S
authors.lastname: Weis
authors.forename: Alexander
authors.initials: A
authors.lastname: Zitzer
authors.forename: Sucharit
authors.initials: S
authors.lastname: Bhakdi
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054353:Perforin; D017354:Point Mutation; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014734:Vibrio cholerae
chemical_list: D004220:Disulfides; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D013438:Sulfhydryl Compounds; D054353:Perforin; C038518:acrylodan; D015081:2-Naphthylamine; D003545:Cysteine