Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel.
The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild-type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.
Masson
L
L
National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec, Canada H4P 2R2. Luke.Masson@Nrc.Ca
Tabashnik
B E
BE
Liu
Y B
YB
Brousseau
R
R
Schwartz
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D004731:Endotoxins / Q000737:chemistry*; D006460:Hemolysin Proteins; D007473:Ion Channels / Q000737:chemistry*; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology; D017433:Protein Structure, Secondary
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007473:Ion Channels; C030956:insecticidal crystal protein, Bacillus Thuringiensis