Topography of diphtheria toxin's T domain in the open channel state

Lisa Senzel, Michael Gordon, Robert O. Blaustein, K. Joon Oh, R. John Collier, Alan Finkelstein

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

When diphtheria toxin encounters a low pH environment, the channel- forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a 'double dagger' model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed 'daggers,' TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ~70 residues, is translocated across the membrane to the trans side.

Original languageEnglish (US)
Pages (from-to)421-434
Number of pages14
JournalJournal of General Physiology
Volume115
Issue number4
DOIs
StatePublished - Apr 2000

Fingerprint

Diphtheria Toxin
Membranes
His-His-His-His-His-His
Streptavidin
Biotin
Cysteine
Catalytic Domain

Keywords

  • Histidine tag
  • Planar bilayers
  • Streptavidin
  • Translocation

ASJC Scopus subject areas

  • Physiology

Cite this

Senzel, L., Gordon, M., Blaustein, R. O., Oh, K. J., Collier, R. J., & Finkelstein, A. (2000). Topography of diphtheria toxin's T domain in the open channel state. Journal of General Physiology, 115(4), 421-434. https://doi.org/10.1085/jgp.115.4.421

Topography of diphtheria toxin's T domain in the open channel state. / Senzel, Lisa; Gordon, Michael; Blaustein, Robert O.; Oh, K. Joon; Collier, R. John; Finkelstein, Alan.

In: Journal of General Physiology, Vol. 115, No. 4, 04.2000, p. 421-434.

Research output: Contribution to journalArticle

Senzel, L, Gordon, M, Blaustein, RO, Oh, KJ, Collier, RJ & Finkelstein, A 2000, 'Topography of diphtheria toxin's T domain in the open channel state', Journal of General Physiology, vol. 115, no. 4, pp. 421-434. https://doi.org/10.1085/jgp.115.4.421
Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A. Topography of diphtheria toxin's T domain in the open channel state. Journal of General Physiology. 2000 Apr;115(4):421-434. https://doi.org/10.1085/jgp.115.4.421
Senzel, Lisa ; Gordon, Michael ; Blaustein, Robert O. ; Oh, K. Joon ; Collier, R. John ; Finkelstein, Alan. / Topography of diphtheria toxin's T domain in the open channel state. In: Journal of General Physiology. 2000 ; Vol. 115, No. 4. pp. 421-434.
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