Insertion and organization within membranes of the δ-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 (alpha 1-alpha 7). According to the "umbrella model," alpha 4 and alpha 5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, alpha 4-loop-alpha 5, its alpha 4 and alpha 5 helices, as well as mutant alpha 4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the alpha 4-loop-alpha 5 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 alpha 4-loop-alpha 5 as the membrane-inserted pore-forming hairpin in which alpha 4 and alpha 5 line the lumen of the channel and alpha 5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active alpha 4 mutant peptide completely inhibits alpha 4-loop-alpha 5 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.