Home LiteratureArticle Details
PMID: 7529493 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

The alpha-5 segment of Bacillus thuringiensis delta-endotoxin: in vitro activity, ion channel formation and molecular modelling.

The Biochemical journal ·Vol. 304 ( Pt 3) ·1994-12-15 ·Pages 895-902

Gazit E, Bach D, Kerr ID, Sansom MS, Chejanovsky N, Shai Y

Abstract

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].

MeSH Terms
4-Chloro-7-nitrobenzofurazan/analogs & derivatives Amino Acid Sequence Animals Bacillus thuringiensis Bacillus thuringiensis Toxins Bacterial Proteins/chemistry,metabolism,toxicity Bacterial Toxins Endotoxins/chemistry,metabolism,toxicity Fluorescence Fluorescent Dyes Hemolysin Proteins Ion Channels/biosynthesis Lipid Bilayers/metabolism Membranes/drug effects Models, Molecular Molecular Sequence Data Protein Structure, Secondary Spodoptera/drug effects
Chemicals
Bacillus thuringiensis Toxins Bacterial Proteins Bacterial Toxins Endotoxins Fluorescent Dyes Hemolysin Proteins Ion Channels Lipid Bilayers insecticidal crystal protein, Bacillus Thuringiensis 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole 4-Chloro-7-nitrobenzofurazan
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Gazit E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bach D
Kerr I D
Sansom M S
Chejanovsky N
Shai Y
References (53)
53 references, click to expand
  1. Synthetic amphiphilic peptide models for protein ion channels.
    Science. 1988 May 27;240(4856):1177-81 PMID: 2453923
  2. Surface, subunit interfaces and interior of oligomeric proteins.
    J Mol Biol. 1988 Nov 5;204(1):155-64 PMID: 3216390
  3. Established insect cell line from the cabbage looper, Trichoplusia ni.
    Nature. 1970 May 2;226(5244):466-7 PMID: 16057320
  4. A molecular blueprint for the pore-forming structure of voltage-gated calcium channels.
    Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6418-22 PMID: 1713679
  5. Structural and functional characterization of the alpha 5 segment of Bacillus thuringiensis delta-endotoxin.
    Biochemistry. 1993 Apr 6;32(13):3429-36 PMID: 8384882
  6. Channel formation properties of synthetic pardaxin and analogues.
    J Biol Chem. 1990 Nov 25;265(33):20202-9 PMID: 1700783
  7. Properties of ion channels formed by Staphylococcus aureus delta-toxin.
    Biochim Biophys Acta. 1988 Jul 21;942(2):280-94 PMID: 2456097
  8. Alpha-helical coiled coils and bundles: how to design an alpha-helical protein.
    Proteins. 1990;7(1):1-15 PMID: 2184436
  9. M2 delta, a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor.
    Proc Natl Acad Sci U S A. 1988 Nov;85(22):8703-7 PMID: 2460876
  10. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis.
    Science. 1990 Jan 5;247(4938):72-4 PMID: 2294593
  11. Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues.
    Biochemistry. 1992 Sep 22;31(37):8868-75 PMID: 1327106
  12. Parallel helix bundles and ion channels: molecular modeling via simulated annealing and restrained molecular dynamics.
    Biophys J. 1994 Oct;67(4):1501-15 PMID: 7529585
  13. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants.
    Science. 1993 Nov 26;262(5138):1401-7 PMID: 8248779
  14. Helix to helix packing in proteins.
    J Mol Biol. 1981 Jan 5;145(1):215-50 PMID: 7265198
  15. Binding and action of cecropin and cecropin analogues: antibacterial peptides from insects.
    Biochim Biophys Acta. 1988 Apr 7;939(2):260-6 PMID: 3128324
  16. Differentiation of lipid-associating helices by use of three-dimensional molecular hydrophobicity potential calculations.
    J Biol Chem. 1991 Aug 25;266(24):16120-7 PMID: 1714906
  17. A mechanism for ion selectivity in potassium channels: computational studies of cation-pi interactions.
    Science. 1993 Sep 24;261(5129):1708-10 PMID: 8378771
  18. Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of a Bacillus thuringiensis delta-endotoxin.
    Mol Membr Biol. 1994 Apr-Jun;11(2):87-92 PMID: 7920867
  19. Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.
    J Biol Chem. 1992 Feb 5;267(4):2311-7 PMID: 1310313
  20. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution.
    Nature. 1991 Oct 31;353(6347):815-21 PMID: 1658659
  21. Automated modeling of coiled coils: application to the GCN4 dimerization region.
    Protein Eng. 1991 Aug;4(6):649-59 PMID: 1946322
  22. The two faces of Bacillus thuringiensis: insecticidal proteins and post-exponential survival.
    Mol Microbiol. 1993 Feb;7(4):489-96 PMID: 8384686
  23. Aggregation and organization of pardaxin in phospholipid membranes. A fluorescence energy transfer study.
    J Biol Chem. 1992 Apr 5;267(10):6502-9 PMID: 1551864
  24. The pore dimensions of gramicidin A.
    Biophys J. 1993 Dec;65(6):2455-60 PMID: 7508762
  25. Membrane fusion activity of succinylated melittin is triggered by protonation of its carboxyl groups.
    Biochemistry. 1987 Jun 30;26(13):4056-62 PMID: 2820482
  26. Molecular model of the action potential sodium channel.
    Proc Natl Acad Sci U S A. 1986 Jan;83(2):508-12 PMID: 2417247
  27. pH-dependent pore formation properties of pardaxin analogues.
    J Biol Chem. 1991 Nov 25;266(33):22346-54 PMID: 1939258
  28. Channel protein engineering: synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers.
    Proc Natl Acad Sci U S A. 1988 Apr;85(7):2393-7 PMID: 2451248
  29. Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins.
    Trends Biochem Sci. 1993 Oct;18(10):391-5 PMID: 8256289
  30. The biophysics of peptide models of ion channels.
    Prog Biophys Mol Biol. 1991;55(3):139-235 PMID: 1715999
  31. The mode of action of Bacillus thuringiensis endotoxins.
    Annu Rev Entomol. 1992;37:615-36 PMID: 1311541
  32. Spectroscopic and functional characterization of the putative transmembrane segment of the minK potassium channel.
    Biochemistry. 1993 Mar 9;32(9):2371-7 PMID: 8443177
  33. Solid-phase synthesis of melittin: purification and functional characterization.
    Biochemistry. 1987 Oct 20;26(21):6627-31 PMID: 3427031
  34. Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin.
    FEMS Microbiol Lett. 1993 Aug 1;111(2-3):255-61 PMID: 8405934
  35. Phospholipid bilayers made from monolayers on patch-clamp pipettes.
    Biophys J. 1983 Aug;43(2):231-6 PMID: 6193818
  36. Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo.
    J Cell Sci. 1983 Mar;60:181-97 PMID: 6874728
  37. The nature of cation-pi binding: interactions between tetramethylammonium ion and benzene in aqueous solution.
    Biophys J. 1993 Jul;65(1):43-7 PMID: 8369448
  38. Location of a Bombyx mori receptor binding region on a Bacillus thuringiensis delta-endotoxin.
    J Biol Chem. 1992 Feb 15;267(5):3115-21 PMID: 1310681
  39. Delta-endotoxins form cation-selective channels in planar lipid bilayers.
    Biochem Biophys Res Commun. 1990 Jun 15;169(2):765-72 PMID: 1694077
  40. Ion channels formed by a highly charged peptide.
    Biochemistry. 1991 Apr 9;30(14):3551-7 PMID: 1707312
  41. Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein.
    FEMS Microbiol Lett. 1990 Mar 1;56(1-2):97-104 PMID: 2332159
  42. Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids.
    Biochemistry. 1987 Jan 13;26(1):39-45 PMID: 3030403
  43. Melittin induces fusion of unilamellar phospholipid vesicles.
    Biochim Biophys Acta. 1983 Aug 10;732(3):668-74 PMID: 6871219
  44. Hydrophilic surface maps of channel-forming peptides: analysis of amphipathic helices.
    Eur Biophys J. 1993;22(4):269-77 PMID: 7504619
  45. Determination of the molecular dynamics of alamethicin using 13C NMR: implications for the mechanism of gating of a voltage-dependent channel.
    Biochemistry. 1992 Jun 9;31(22):5136-44 PMID: 1606136
  46. Location of the Bombyx mori specificity domain on a Bacillus thuringiensis delta-endotoxin protein.
    Proc Natl Acad Sci U S A. 1989 Jun;86(11):4037-41 PMID: 2542961
  47. Insecticidal crystal proteins of Bacillus thuringiensis.
    Microbiol Rev. 1989 Jun;53(2):242-55 PMID: 2666844
  48. Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study.
    Biochim Biophys Acta. 1991 Mar 18;1063(1):36-44 PMID: 1707671
  49. Early response of cultured lepidopteran cells to exposure to delta-endotoxin from Bacillus thuringiensis: involvement of calcium and anionic channels.
    Biochim Biophys Acta. 1991 Jun 18;1065(2):250-60 PMID: 1711900
  50. Interaction of D-amino acid incorporated analogues of pardaxin with membranes.
    Biochemistry. 1992 Oct 6;31(39):9482-90 PMID: 1390731
  51. Synthesis, location, and lateral mobility of fluorescently labeled ubiquinone 10 in mitochondrial and artificial membranes.
    Biochemistry. 1989 Apr 18;28(8):3168-76 PMID: 2742832
  52. A three-dimensional molecular assembly model of a lipoprotein from the Escherichia coli outer membrane.
    Proc Natl Acad Sci U S A. 1974 Jun;71(6):2396-2400 PMID: 4601588
  53. The structure of the voltage-sensitive sodium channel. Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure.
    FEBS Lett. 1985 Dec 2;193(2):125-34 PMID: 2415395
Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj3040895
Published
1994-12-15
Pages
895-902
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1137417
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1042/bj3040895 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2