The assembly and organization of the α5 and α7 helices from the pore-forming domain of Bacillus thuringiensis δ-endotoxin: Relevance to a functional model

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA δ-endotoxin contains two helices, α5 and α7, that are highly conserved within all different Cry 5-endotoxins. To gain information on the mode of action of δ-endotoxins, we have used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability to self-assemble and to co-assemble within lipid membranes of α5 and α7. Circular dichroism (CD) spectroscopy revealed that α7 adopts a predominantly α-helical structure in methanol, similar to what has been found for α5, and consistent with its structure in the intact molecule. The hydrophobic moment of α7 is higher than that calculated for α5; however, α7 has a lesser ability to permeate phospholipids as compared to α5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that α7 binds to phospholipid vesicles with a partition coefficient in the order of 10⁴ M^-1 similar to α5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with α5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, α5 self-associates but α7 does not, and that α5 coassembles with α7 but not with an unrelated membrane bound α-helical peptide. Furthermore, resonance energy transfer experiments, using α5 segments, specifically labeled in either the N-or C-terminal sides, suggest a parallel organization of α5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of δ-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where α5 has transmembrane localization and may be part of the pore lining segment(s) while α7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.