Cysteine scanning mutagenesis of alpha4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa.
Helix alpha4 of Bacillus thuringiensis Cry toxins is thought to line the lumen of the pores they form in the midgut epithelial cells of susceptible insect larvae. To define its functional role in pore formation, most of the alpha4 amino acid residues were replaced individually by a cysteine in the Cry1Aa toxin. The toxicities and pore-forming abilities of the mutated toxins were examined, respectively, by bioassays using neonate Manduca sexta larvae and by a light-scattering assay using midgut brush border membrane vesicles isolated from M. sexta. A majority of these mutants had considerably reduced toxicities and pore-forming abilities. Most mutations causing substantial or complete loss of activity map on the hydrophilic face of the helix, while most of those having little or only relatively minor effects map on its hydrophobic face. The properties of the pores formed by mutants that retain significant activity appear similar to those of the pores formed by the wild-type toxin, suggesting that mutations resulting in a loss of activity interfere mainly with pore formation.
Girard
Frédéric
F
Groupe d'Etude des Protéines Membranaires, Université de Montréal, P.O. Box 6128, Centre Ville Station, Montreal, Quebec H3C 3J7, Canada. raynald.laprade@umontreal.ca
Vachon
Vincent
V
Préfontaine
Gabrielle
G
Marceau
Lucie
L
Su
Yanhui
Y
Larouche
Geneviève
G
Vincent
Charles
C
Schwartz
Jean-Louis
JL
Masson
Luke
L
Laprade
Raynald
R
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000502:physiology*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D001835:Body Weight; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D007814:Larva / Q000187:drug effects; D018402:Manduca / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D010539:Permeability; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D017434:Protein Structure, Tertiary; D016019:Survival Analysis
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D052899:Pore Forming Cytotoxic Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis