Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain

Jared A. Silvermani, Joseph A. Mindell, Alan Finkelstein, Wei Hai Shent, R. John Collier

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74 Scopus citations

Abstract

Entry of the catalytic domain of diphtheria toxin into the cytoplasm of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH. To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain. Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K. Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin. Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells >100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction. All three mutations also altered the pH- dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane. E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5. Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain. A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action. The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model. The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain.

Original languageEnglish (US)
Pages (from-to)22524-22532
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number36
StatePublished - Sep 9 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Silvermani, J. A., Mindell, J. A., Finkelstein, A., Shent, W. H., & Collier, R. J. (1994). Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain. Journal of Biological Chemistry, 269(36), 22524-22532.