Weizmann Institute of Science, Rehovot 76100, Israel
bmshai@Weizmann.Weizmann.ac.il
The insecticidal d-endotoxins are produced by Bacillus thuringiensis (B.t.) bacteria during sporulation. The toxins are assumed to act via the formation of transmembrane pores in the midguts of the susceptible insects. The X-ray structure of CryIIIA toxin in aqueous solution was resolved [Li, J., Carroll, J., and Ellar, D. J. (1991) Nature 353, 815-821] and revealed a pore forming domain, which is composed of seven alpha-helices, termed alpha1-alpha7. Nevertheless, the structure and the organization state of the toxin within the membrane environment remains elusive.
We utilized a synthetic peptide approach combined with various spectroscopic methods to investigate the structure and organization of the protein within the membrane. Peptides with sequences corresponding to all of the seven helices of the toxin were synthesized and characterized. The interaction of the peptides with phospholipid membranes was monitored using their environmentally-sensitive fluorescent derivatives. Six of the helices, helices alpha2-alpha7, were found to bind phospholipid vesicles with high affinity, which suggests that they have a structural role in the pores formed by the toxin. The structure of the helices and their orientation relative to the normal of phospholipid membranes, was studied by using attenuated total reflectance furrier transform infrared (ATR FT-IR) spectroscopy. The frequencies of the amide I and II absorption peaks of the peptides reconstituted into lipid multybilayers, indicated that the peptides are predominantly alpha-helical. The ATR dichroic ratio of the amide I bands of the peptides reconstituted to oriented phospholipid membranes, indicated different orientations for the various peptides. Interestingly, only alpha4 and alpha5 were preferentially oriented in a transmembrane orientation, while the rest of the helices were oriented randomly or nearly parallel to the surface of the lipid membranes. Finally, resonance energy transfer experiments between donor/acceptor-labeled segments revealed specificities in their interactions within the membrane.
Taken together, our results are consistent with an "umbrella" model for the structure of the pores formed by the toxin. This model involves the insertion into the membrane of two segments as an helical hairpin, whereas the other helices are lying on the surface of the membrane like the ribs of an umbrella.