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 prote in 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 synthe sized 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 mutatio ns 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 i nto membranes. Together with spectrofluorometric studies, we provide direct evidence for the role of alpha 4-loop-alpha 5 as the membrane-inserted por e-forming hairpin in which alpha 4 and alpha 5 line the lumen of the channe l and alpha 5 also participates in the oligomerization of the toxin. Striki ngly, 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 "immun ity protein" by directly interacting with the segments that form the pore. This presents a potential means of interfering with the assembly and functi on of other membrane proteins as well.