The Assembly and Organization of the α5 and α7 Helices from the Pore-forming Domain of Bacillusthuringiensis δ-Endotoxin: RELEVANCE TO A FUNCTIONAL MODEL

Abstract

The pore-forming domain of Bacillus thuringiensis insecticidal CryIIIA delta-endotoxin contains two helices, alpha 5 and alpha 7, that are highly conserved within all different Cry delta-endotoxins. To gain information on the mode of action of delta-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 alpha 5 and alpha 7. Circular dichroism (CD) spectroscopy revealed that alpha 7 adopts a predominantly alpha-helical structure in methanol, similar to what has been found for alpha 5, and consistent with its structure in the intact molecule. The hydrophobic moment of alpha 7 is higher than that calculated for alpha 5; however, alpha 7 has a lesser ability to permeate phospholipids as compared to alpha 5. Binding experiments with 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labeled peptide demonstrated that alpha 7 binds to phospholipid vesicles with a partition coefficient in the order of 10(4) M-1 similar to alpha 5, but with reduced kinetics and in a noncooperative manner, as opposed to the fast kinetics and cooperativity found with alpha 5. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that, in their membrane-bound state, alpha 5 self-associates but alpha 7 does not, and that alpha 5 coassembles with alpha 7 but not with an unrelated membrane bound alpha-helical peptide. Furthermore, resonance energy transfer experiments, using alpha 5 segments, specifically labeled in either the N- or C-terminal sides, suggest a parallel organization of alpha 5 monomers within the membranes. Taken together the results are consistent with an umbrella model suggested for the pore forming activity of delta-endotoxin (Li, J., Caroll, J., and Ellar, D. J. (1991) Nature 353, 815-821), where alpha 5 has transmembrane localization and may be part of the pore lining segment(s) while alpha 7 may serve as a binding sensor that initiates the binding of the pore domain to the membrane.