title: Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1Aa.
abstract: Helix 3 of the Cry1Aa toxin from Bacillus thuringiensis possesses eight charged amino acids. These residues, with the exception of those involved in intramolecular salt bridges (E90, R93, E112, and R115), were mutated individually either to a neutral or to an oppositely charged amino acid. The mutated genes were expressed, and the resultant, trypsin-activated toxins were assessed for their toxicity to Manduca sexta larvae and their ability to permeabilize M. sexta larval midgut brush border membrane vesicles to KCl, sucrose, raffinose, potassium gluconate, and N-methyl-D-glucamine hydrochloride with a light-scattering assay based on osmotic swelling. Most mutants were considerably less toxic than Cry1Aa. Replacing either E101, E116, E118, or D120 by cysteine, glutamine, or lysine residues had only minor effects on the properties of the pores formed by the modified toxins. However, half of these mutants (E101C, E101Q, E101K, E116K, E118C, and D120K) had a significantly slower rate of pore formation than Cry1Aa. Mutations at R99 (R99C, R99E, and R99Y) resulted in an almost complete loss of pore-forming ability. These results are consistent with a model in which alpha-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.
authors: Groupe de recherche en transport membranaire, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec, H3C 3J7, Canada.
authors: Vincent
authors: V
authors: Vachon
authors: Gabrielle
authors: G
authors: Préfontaine
authors: Florence
authors: F
authors: Coux
authors: Cécile
authors: C
authors: Rang
authors: Lucie
authors: L
authors: Marceau
authors: Luke
authors: L
authors: Masson
authors: Roland
authors: R
authors: Brousseau
authors: Roger
authors: R
authors: Frutos
authors: Jean-Louis
authors: JL
authors: Schwartz
authors: Raynald
authors: R
authors: Laprade
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001427:Bacterial Toxins; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D006460:Hemolysin Proteins; D007700:Kinetics; D018402:Manduca / Q000187:drug effects / Q000378:metabolism; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis