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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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The nature of the streptavidin-induced noise. Before the start of the record, T domain biotinylated at residue 261 was added to the cis compartment to a concentration of ∼50 ng/ml. During the entire experiment, the voltage was held at +60 mV, except for occasional pulses to 0 mV during the breaks. Conductance increased linearly for several minutes. Before the first arrow, it was 467 pS, corresponding to ∼12 channels. At the first arrow (during a 25-s break), streptavidin was added to the trans compartment to a concentration of 30 μg/ml. Immediately after streptavidin addition, the amount of noise increased significantly, and the conductance increased slowly to ∼1.4 nS, corresponding to 35 channels. Note that the noise represents fluctuations to higher, not lower, conductances. During the second break, lasting 4.5 min, conductance continued to rise slowly, to a level of 1.5 nS, corresponding to ∼37 channels. TCEP, pH 7.1, was then added to the trans compartment to a concentration of 20 mM. Subsequently, the conductance fell to the level seen after the break, and the noise decreased drastically. The connected arrows show points in the record of equal conductance (∼32 channels) before and after TCEP addition; note the difference in noise at these points. The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 5 mM Mes, pH 5.3, and the trans contained 5 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
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Figure 8: The nature of the streptavidin-induced noise. Before the start of the record, T domain biotinylated at residue 261 was added to the cis compartment to a concentration of ∼50 ng/ml. During the entire experiment, the voltage was held at +60 mV, except for occasional pulses to 0 mV during the breaks. Conductance increased linearly for several minutes. Before the first arrow, it was 467 pS, corresponding to ∼12 channels. At the first arrow (during a 25-s break), streptavidin was added to the trans compartment to a concentration of 30 μg/ml. Immediately after streptavidin addition, the amount of noise increased significantly, and the conductance increased slowly to ∼1.4 nS, corresponding to 35 channels. Note that the noise represents fluctuations to higher, not lower, conductances. During the second break, lasting 4.5 min, conductance continued to rise slowly, to a level of 1.5 nS, corresponding to ∼37 channels. TCEP, pH 7.1, was then added to the trans compartment to a concentration of 20 mM. Subsequently, the conductance fell to the level seen after the break, and the noise decreased drastically. The connected arrows show points in the record of equal conductance (∼32 channels) before and after TCEP addition; note the difference in noise at these points. The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 5 mM Mes, pH 5.3, and the trans contained 5 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.

Mentions: The cis and trans streptavidin-induced increases in macroscopic current noise described above were not reflected in an increase in single-channel current noise, but were seen with as few as 12 channels present (Fig. 8). At this level, it was apparent that streptavidin induced current flickerings to larger, but not to smaller, values (Fig. 8). Thus, streptavidin's effect on current noise does not result from its transiently blocking channels, in which case increased flickering closures should have occurred. Whatever the mechanism, the streptavidin assay clearly locates residues 235, 261, and 267 on the trans side and residues 291 and 376 on the cis side, in agreement with the findings from the chemically attached H6 peptide assay.


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)

The nature of the streptavidin-induced noise. Before the start of the record, T domain biotinylated at residue 261 was added to the cis compartment to a concentration of ∼50 ng/ml. During the entire experiment, the voltage was held at +60 mV, except for occasional pulses to 0 mV during the breaks. Conductance increased linearly for several minutes. Before the first arrow, it was 467 pS, corresponding to ∼12 channels. At the first arrow (during a 25-s break), streptavidin was added to the trans compartment to a concentration of 30 μg/ml. Immediately after streptavidin addition, the amount of noise increased significantly, and the conductance increased slowly to ∼1.4 nS, corresponding to 35 channels. Note that the noise represents fluctuations to higher, not lower, conductances. During the second break, lasting 4.5 min, conductance continued to rise slowly, to a level of 1.5 nS, corresponding to ∼37 channels. TCEP, pH 7.1, was then added to the trans compartment to a concentration of 20 mM. Subsequently, the conductance fell to the level seen after the break, and the noise decreased drastically. The connected arrows show points in the record of equal conductance (∼32 channels) before and after TCEP addition; note the difference in noise at these points. The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 5 mM Mes, pH 5.3, and the trans contained 5 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
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Related In: Results  -  Collection

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Figure 8: The nature of the streptavidin-induced noise. Before the start of the record, T domain biotinylated at residue 261 was added to the cis compartment to a concentration of ∼50 ng/ml. During the entire experiment, the voltage was held at +60 mV, except for occasional pulses to 0 mV during the breaks. Conductance increased linearly for several minutes. Before the first arrow, it was 467 pS, corresponding to ∼12 channels. At the first arrow (during a 25-s break), streptavidin was added to the trans compartment to a concentration of 30 μg/ml. Immediately after streptavidin addition, the amount of noise increased significantly, and the conductance increased slowly to ∼1.4 nS, corresponding to 35 channels. Note that the noise represents fluctuations to higher, not lower, conductances. During the second break, lasting 4.5 min, conductance continued to rise slowly, to a level of 1.5 nS, corresponding to ∼37 channels. TCEP, pH 7.1, was then added to the trans compartment to a concentration of 20 mM. Subsequently, the conductance fell to the level seen after the break, and the noise decreased drastically. The connected arrows show points in the record of equal conductance (∼32 channels) before and after TCEP addition; note the difference in noise at these points. The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 5 mM Mes, pH 5.3, and the trans contained 5 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
Mentions: The cis and trans streptavidin-induced increases in macroscopic current noise described above were not reflected in an increase in single-channel current noise, but were seen with as few as 12 channels present (Fig. 8). At this level, it was apparent that streptavidin induced current flickerings to larger, but not to smaller, values (Fig. 8). Thus, streptavidin's effect on current noise does not result from its transiently blocking channels, in which case increased flickering closures should have occurred. Whatever the mechanism, the streptavidin assay clearly locates residues 235, 261, and 267 on the trans side and residues 291 and 376 on the cis side, in agreement with the findings from the chemically attached H6 peptide assay.

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