The alpha-5 segment of Bacillus thuringiensis delta-endotoxin: in vitro activity, ion channel formation and molecular modelling.
A peptide with a sequence corresponding to the highly conserved alpha-5 segment of the Cry delta-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA [Gazit and Shai (1993) Biochemistry 32, 3429-3436]), 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 alpha-5 at either the N-terminal amino acid or the epsilon-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 alpha-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 alpha-5 peptides were protected from enzymic proteolysis. Functional characterization of alpha-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 alpha-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 alpha-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support 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 toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB delta-endotoxin in a loop between alpha-5 and alpha-6 is highly important for its larvicidal activity [Angsuthanasombat, Crickmore and Ellar (1993) FEMS Microbiol. Lett. 111, 255-262].
Gazit
E
E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bach
D
D
Kerr
I D
ID
Sansom
M S
MS
Chejanovsky
N
N
Shai
Y
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007473:Ion Channels / Q000096:biosynthesis*; D008051:Lipid Bilayers / Q000378:metabolism; D008566:Membranes / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D018411:Spodoptera / Q000187:drug effects
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D005456:Fluorescent Dyes; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; C030956:insecticidal crystal protein, Bacillus Thuringiensis; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan