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 α-helix bundle (α1-α7). According to the 'umbrella model,' α4 and α5 helices form a hairpin structure thought to be inserted into the membrane upon binding. Here, we have synthesized and characterized the hairpin domain, α4-loop-α5, its α4 and α5 helices, as well as mutant α4 peptides based on mutations that increased or decreased toxin toxicity. Membrane permeation studies revealed that the α4-loop-α5 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 α4-loop-α5 as the membrane-inserted pore-forming hairpin in which α4 and α5 line the lumen of the channel and α5 also participates in the oligomerization of the toxin. Strikingly, the addition of the active α4 mutant peptide completely inhibits α-loop-α5 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.