title: Chemical modification of Bacillus thuringiensis Cry1Aa toxin single-cysteine mutants reveals the importance of domain I structural elements in the mechanism of pore formation.
abstract: Bacillus thuringiensis Cry toxins form pores in the apical membrane of insect larval midgut cells. To investigate their mechanism of membrane insertion, mutants in which cysteine replaced individual amino acids located within the pore-forming domain of Cry1Aa were chemically modified with sulfhydryl-specific reagents. The thiol group of cysteine was highly susceptible to oxidation and its reactivity was significantly increased when the toxins were purified under reducing conditions. Addition of a biotin group to the cysteine had little effect on the ability of the toxins to permeabilize Manduca sexta brush border membrane vesicles except for a slight reduction in activity for S252C and a large increase in activity for Y153C. The activity of Y153C was also significantly increased after modification by reagents that added an aromatic or a charged group to the cysteine. When permeability assays were performed in the presence of streptavidin, a large biotin-binding protein, the pore-forming activity of several mutants, including Y153C, where the altered residue is located within the hairpin comprising helices alpha4 and alpha5, or in adjacent loops, was significantly reduced. These results support the umbrella model of toxin insertion.
authors.affiliation: Groupe d'étude des protéines membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, Canada H3C 3J7.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Girard
authors.forename: Vincent
authors.initials: V
authors.lastname: Vachon
authors.forename: Geneviève
authors.initials: G
authors.lastname: Lebel
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Préfontaine
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Schwartz
authors.forename: Luke
authors.initials: L
authors.lastname: Masson
authors.forename: Raynald
authors.initials: R
authors.lastname: Laprade
mesh_terms: D001413:Bacillus thuringiensis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D016062:Porosity; D011487:Protein Conformation
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D003545:Cysteine