title: Effects of mutations within surface-exposed loops in the pore-forming domain of the Cry9Ca insecticidal toxin of Bacillus thuringiensis.
abstract: The pore-forming domain of Bacillus thuringiensis insecticidal Cry toxins is formed of seven amphipathic α-helices. Because pore formation is thought to involve conformational changes within this domain, the possible role of its interhelical loops in this crucial step was investigated with Cry9Ca double mutants, which all share the previously characterized R164A mutation, using a combination of homology modeling, bioassays and electrophysiological measurements. The mutations either introduced, neutralized or reversed an electrical charge carried by a single residue of one of the domain I loops. The ability of the 28 Cry9Ca double mutants to depolarize the apical membrane of freshly isolated Manduca sexta larval midguts was tested in the presence of either midgut juice or a cocktail of protease inhibitors because these conditions had been shown earlier to greatly enhance pore formation by Cry9Ca and its R164A single-site mutant. Most mutants retained toxicity toward neonate larvae and a pore-forming ability in the electrophysiological assay, which were comparable to those of their parental toxin. In contrast, mutants F130D, L186D and V189D were very poorly toxic and practically inactive in vitro. On the other hand, mutant E129A depolarized the midgut membrane efficiently despite a considerably reduced toxicity, and mutant Q192E displayed a reduced depolarizing ability while conserving a near wild-type toxicity. These results suggest that the conditions found in the insect midgut, including high ionic strength, contribute to minimizing the influence of surface charges on the ability of Cry9Ca and probably other B. thuringiensis toxins to form pores within their target membrane.
authors.affiliation: Groupe d'étude des protéines membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, QC, H3C 3J7, Canada. jean-frederic.brunet@umontreal.ca
authors.forename: Jean-Frédéric
authors.initials: JF
authors.lastname: Brunet
authors.forename: Vincent
authors.initials: V
authors.lastname: Vachon
authors.forename: Mireille
authors.initials: M
authors.lastname: Marsolais
authors.forename: Greta
authors.initials: G
authors.lastname: Arnaut
authors.forename: Jeroen
authors.initials: J
authors.lastname: Van Rie
authors.forename: Lucie
authors.initials: L
authors.lastname: Marceau
authors.forename: Geneviève
authors.initials: G
authors.lastname: Larouche
authors.forename: Charles
authors.initials: C
authors.lastname: Vincent
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Schwartz
authors.forename: Raynald
authors.initials: R
authors.lastname: Laprade
mesh_terms: D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D004594:Electrophysiology; D004731:Endotoxins / Q000235:genetics* / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics* / Q000494:pharmacology; D066298:In Vitro Techniques; D007814:Larva / Q000187:drug effects; D018402:Manduca / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis