Gazit, E., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
Bach, D., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
Kerr, I.D., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
Sansom, M.S.P., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
Chejanovsky, N., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel
Shai, Y., Dept. Membrane Research Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel

A peptide with a sequence corresponding to the highly conserved α-5 segment of the Cry δ-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA, was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of α-5 at either the N-terminal amino acid or the ε-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled α-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled α-5 peptides were protected from enzymic proteolysis. Functional characterization of α-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of α-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by α-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for α-5 in the toxic mechanism of δ-endotoxins, and assign α-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB δ-endotoxin in a loop between α-5 and α-6 is highly important for its larvicidal activity.