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

Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2000)

Bottom Line: We find that there are three membrane-spanning segments.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 approximately 70 residues, is translocated across the membrane to the trans side.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

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 approximately 70 residues, is translocated across the membrane to the trans side.

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(Top) Linear diagram of the whole diphtheria toxin (DT) molecule (protease nicked), with an expanded view of the channel-forming T domain. Black regions denote hydrophobic stretches within the T domain. (Bottom) Sequence of the H6-tagged T domain, slightly modified from Senzel et al. 1998 and edited to correct two errors in that sequence.
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Figure 1: (Top) Linear diagram of the whole diphtheria toxin (DT) molecule (protease nicked), with an expanded view of the channel-forming T domain. Black regions denote hydrophobic stretches within the T domain. (Bottom) Sequence of the H6-tagged T domain, slightly modified from Senzel et al. 1998 and edited to correct two errors in that sequence.

Mentions: Diphtheria toxin is a single, 535-residue polypeptide containing three domains: the amino-terminal C, or catalytic, domain (residues 1–185); the carboxy-terminal R, or receptor-binding, domain (residues 386–535), and the translocation, or T domain, lying between them (residues 202–378) (Fig. 1). The catalytic domain is connected to the T domain by a protease-susceptible loop and by an easily reducible disulfide bridge.


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

Senzel L, Gordon M, Blaustein RO, Oh KJ, Collier RJ, Finkelstein A - J. Gen. Physiol. (2000)

(Top) Linear diagram of the whole diphtheria toxin (DT) molecule (protease nicked), with an expanded view of the channel-forming T domain. Black regions denote hydrophobic stretches within the T domain. (Bottom) Sequence of the H6-tagged T domain, slightly modified from Senzel et al. 1998 and edited to correct two errors in that sequence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233753&req=5

Figure 1: (Top) Linear diagram of the whole diphtheria toxin (DT) molecule (protease nicked), with an expanded view of the channel-forming T domain. Black regions denote hydrophobic stretches within the T domain. (Bottom) Sequence of the H6-tagged T domain, slightly modified from Senzel et al. 1998 and edited to correct two errors in that sequence.
Mentions: Diphtheria toxin is a single, 535-residue polypeptide containing three domains: the amino-terminal C, or catalytic, domain (residues 1–185); the carboxy-terminal R, or receptor-binding, domain (residues 386–535), and the translocation, or T domain, lying between them (residues 202–378) (Fig. 1). The catalytic domain is connected to the T domain by a protease-susceptible loop and by an easily reducible disulfide bridge.

Bottom Line: We find that there are three membrane-spanning segments.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 approximately 70 residues, is translocated across the membrane to the trans side.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.

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 approximately 70 residues, is translocated across the membrane to the trans side.

Show MeSH
Related in: MedlinePlus