Role of Helix 3 in Pore Formation by the Bacillus thuringiensis Insecticidal Toxin Cry1Aa
- 17 April 2002
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 41 (19) , 6178-6184
- https://doi.org/10.1021/bi011572e
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 α-helix 3 plays an important role in the mechanism of pore formation without being directly involved in determining the properties of the pores.Keywords
This publication has 9 references indexed in Scilit:
- Helix 4 of the Bacillus thuringiensis Cry1Aa Toxin Lines the Lumen of the Ion ChannelJournal of Biological Chemistry, 1999
- Amino acid substitution in α‐helix 7 of Cry1Ac δ‐endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera HubnerFEBS Letters, 1999
- Bacillus thuringiensis and Its Pesticidal Crystal ProteinsMicrobiology and Molecular Biology Reviews, 1998
- Simulation of voltage-dependent interactions of α-helical peptides with lipid bilayersBiophysical Chemistry, 1996
- Bacillus thuringiensisCrylA(a) Insecticidal Toxin: Crystal Structure and Channel FormationJournal of Molecular Biology, 1995
- Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin geneJournal of Bacteriology, 1995
- The α-5 segment of Bacillus thuringiensis δ-endotoxin: in vitro activity, ion channel formation and molecular modellingBiochemical Journal, 1994
- Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of aBacillus thuringiensisδ-endotoxinMolecular Membrane Biology, 1994
- An analysis of Bacillus thuringiensisδ‐endotoxin action on insect‐midgut‐membrane permeability using a light‐scattering assayEuropean Journal of Biochemistry, 1993