Helix 4 of the Bacillus thuringiensis Cry1Aa toxin lines the lumen of the ion channel

Luke Masson, Bruce E. Tabashnik, Liu Yong-Biao, Roland Brousseau, Jean Louis Schwartz

Research output: Contribution to journalArticle

99 Scopus citations

Abstract

The mode of action of Bacillus thuringiensis insecticidal proteins is not well understood. Based on analogies with other bacterial toxins and ion channels, we hypothesized that charged amino acids in helix 4 of the Cry1Aa toxin are critical for toxicity and ion channel function. Using Plutella xylostella as a model target, we analyzed responses to Cry1Aa and eight proteins with altered helix 4 residues. Toxicity was abolished in five charged residue mutants (E129K, R131Q, R131D, D136N, D136C), however, two charged (R127E and R127N) and one polar (N138C) residue mutant retained wild- type toxicity. Compared with Cry1Aa and toxic mutants, nontoxic mutants did not show greatly reduced binding to brush border membrane vesicles, but their ion channel conductance was greatly reduced in planar lipid bilayers. Substituted cysteine accessibility tests showed that in situ restoration of the negative charge of D136C restored conductance to wild-type levels. The results imply that charged amino acids on the Asp-136 side of helix 4 are essential for toxicity and passage of ions through the channel. These results also support a refined version of the umbrella model of membrane integration in which the side of helix 4 containing Asp-136 faces the aqueous lumen of the ion channel.

Original languageEnglish (US)
Pages (from-to)31996-32000
Number of pages5
JournalJournal of Biological Chemistry
Volume274
Issue number45
DOIs
StatePublished - Nov 5 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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