Insertion and organization within membranes of the delta-endotoxin pore-forming domain, helix 4-loop-helix 5, and inhibition of its activity by a mutant helix 4 peptide.
The pore-forming domain of Bacillus thuringiensis Cry1Ac insecticidal protein comprises of a seven alpha-helix bundle (alpha1-alpha7). According to the "umbrella model," alpha4 and alpha5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha4-loop-alpha5, its alpha4 and alpha5 helices, as well as mutant alpha4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha4-loop-alpha5 hairpin is extremely active compared with the isolated helices or their mixtures, indicating the complementary role of the two helices and the need for the loop for efficient insertion into membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha4-loop-alpha5 as the membrane-inserted pore-forming hairpin in which alpha4 and alpha5 line the lumen of the channel and alpha5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha4 mutant peptide completely inhibits alpha4-loop-alpha5 pore formation, thus providing, to our knowledge, the first example that a mutated helix within a pore can function as an "immunity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and function of other membrane proteins as well.
Gerber
D
D
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Shai
Y
Y
D000595:Amino Acid Sequence; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004731:Endotoxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006460:Hemolysin Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010539:Permeability; D018272:Porins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D018272:Porins; C030956:insecticidal crystal protein, Bacillus Thuringiensis