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. 2001 Dec;67(12):5715-20.
doi: 10.1128/AEM.67.12.5715-5720.2001.

The alpha-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins

N J Tigue  1 , J Jacoby, D J Ellar
Affiliations

The alpha-helix 4 residue, Asn135, is involved in the oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins

N J Tigue et al. Appl Environ Microbiol. 2001 Dec.

Abstract

The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.

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Figures

FIG. 1
FIG. 1
Extent of volume recovery obtained by M. sexta BBMV when incubated with increasing amounts of wild-type toxins (in picomoles per milligram of BBMV: solid triangles, 30; open triangles, 60; open diamonds, 150; solid squares, 300; open squares, 600; solid circles, 900). Not every concentration was used for both toxins. Only one concentration of each N135Q mutant is illustrated (open circles, 1,500 pmol/mg of BBMV). (A) Cry1Ac1 and Cry1Ac1N135Q. (B) Cry1Ab5 and its NQ mutant.
FIG. 2
FIG. 2
Dissociation of 125I-labeled wild-type and mutant toxins from M. sexta BBMV. The amount of binding is expressed as a percentage of the toxin bound following the addition of an excess of the equivalent unlabeled toxin. Nonspecific binding was subtracted from total binding. (A) Solid squares, Cry1Ac1; open circles, Cry1Ac1 N135Q. (B) Shaded squares, Cry1Ab5; open circles, Cry1Ab5 N135Q.
FIG. 3
FIG. 3
Immunoblots illustrating the oligomerization state of wild-type and mutant toxin molecules bound to M. sexta membrane vesicles. (A) Cry1Ac1. (B) Cry1Ac1NQ. (C) Cry1Ab5. (D) Cry1Ab5NQ. M, markers; T, toxin only; P, pellet fraction; S, supernatant.
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References

    1. Ahmad W, Ellar D J. Directed mutagenesis of selected regions of a Bacillus thuringiensis entomocidal protein. FEMS Microbiol Lett. 1990;68:97–104. - PubMed
    1. Aronson A I, Geng C, Wu L. Aggregation of Bacillus thuringiensis Cry1A toxins upon binding to target insect larval midgut vesicles. Appl Environ Microbiol. 1999;65:2503–2507. - PMC - PubMed
    1. Bell R A, Joachim F G. Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms. Ann Entomol Soc Am. 1976;69:365–373.
    1. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. - PubMed
    1. Burton S L, Ellar D J, Li J, Derbyshire D J. N-Acetylgalactosamine on the putative insect receptor aminopeptidase N is recognised by a site on the domain III lectin-like fold of a Bacillus thuringiensis insecticidal toxin. J Mol Biol. 1999;287:1011–1022. - PubMed

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