THE ALPHA-5 SEGMENT OF BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN - IN-VITRO ACTIVITY, ION-CHANNEL FORMATION AND MOLECULAR MODELING

A peptide with a sequence corresponding to the highly conserved alpha- 5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Ba cillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3 429-3436]), was investigated with respect to its interaction with inse ct membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf- 9) cells, and its propensity to form ion channels in planar lipid memb ranes (PLMs). Selectively labelled analogues of alpha-5 at either the N-terminal animo acid or the epsilon-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fl uorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl ( NBD)-labelled alpha-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the reloc ation of the fluorescent probes to an environment of increased apolari ty, i.e. within the lipidic constituent of the membrane. Moreover, mid gut membrane-bound NBD-labelled alpha-5 peptides were protected from e nzymic proteolysis. Functional characterization of alpha-5 has reveale d that it is cytotoxic to Sf-9 insect cells, and that it forms ion cha nnels in PLMs with conductances ranging from 30 to 1000 pS. A proline- substituted analogue of alpha-5 is less cytolytic and slightly more ex posed to enzymic digestion. Molecular modelling utilizing simulated an nealing via molecular dynamics suggests that a transbilayer pore may b e formed by alpha-5 monomers that assemble to form a left-handed coile d coil of approximately parallel helices. These findings further suppo rt a role for alpha-5 in the toxic mechanism, of delta-endotoxins, and assign alpha-5 as one of the transmembrane helices which form the tox ic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alph a-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].