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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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H6 peptide chemically linked at residues 293 or 320 blocks the T domain channel at positive voltages. Before the start of each record, T domain with H6 peptide attached at residue 293 (A) or 320 (B) was added to the cis compartment to a concentration of ∼300 (A) or 400 (B) ng/ml. (A) After addition of the T domain, conductance rose over time to the level seen at the start of the record, with the voltage held at +20 mV. When the voltage was switched to −20 mV, the conductance nearly doubled. Subsequent voltage steps to +30, +40, and +50 mV with intervening pulses to corresponding negative voltages demonstrated two facts. (a) The conductance at each positive test voltage was less than that at the corresponding negative voltage; the magnitude of the rectification increased with larger voltages. (b) Positive 10-mV increments produced disproportionately small current increases. (B) A single channel flickered between the open state and a zero-conductance blocked state at +60 mV, spending almost all its time in the zero-conductance state. When the voltage was switched to −60 mV, the channel became unblocked. Similar behavior was seen at ±80 mV after a 1-min break. (Records similar to those in A and B were obtained with T domain having the H6 peptide attached at residue 376, 291, or 294.) The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 30 mM Mes, pH 5.3, and the trans contained 50 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
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Figure 5: H6 peptide chemically linked at residues 293 or 320 blocks the T domain channel at positive voltages. Before the start of each record, T domain with H6 peptide attached at residue 293 (A) or 320 (B) was added to the cis compartment to a concentration of ∼300 (A) or 400 (B) ng/ml. (A) After addition of the T domain, conductance rose over time to the level seen at the start of the record, with the voltage held at +20 mV. When the voltage was switched to −20 mV, the conductance nearly doubled. Subsequent voltage steps to +30, +40, and +50 mV with intervening pulses to corresponding negative voltages demonstrated two facts. (a) The conductance at each positive test voltage was less than that at the corresponding negative voltage; the magnitude of the rectification increased with larger voltages. (b) Positive 10-mV increments produced disproportionately small current increases. (B) A single channel flickered between the open state and a zero-conductance blocked state at +60 mV, spending almost all its time in the zero-conductance state. When the voltage was switched to −60 mV, the channel became unblocked. Similar behavior was seen at ±80 mV after a 1-min break. (Records similar to those in A and B were obtained with T domain having the H6 peptide attached at residue 376, 291, or 294.) The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 30 mM Mes, pH 5.3, and the trans contained 50 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.

Mentions: When the synthetic H6 peptide was tethered to either residue 291, 293, 294, 320, or 376, the T domain conductance declined very rapidly at positive voltages and increased very rapidly at negative voltages (Fig. 5 A). (The transients were too fast to be recorded at our 100-Hz filtering.) At the single-channel level, the channels spent most of their time in a zero-conductance blocked state at +60 or +80 mV; when the sign of the voltage was reversed, the channels recovered immediately to the open state (Fig. 5 B). Thus, at both the macroscopic and single-channel levels, channels with tethered H6 peptide at these residues qualitatively displayed the same voltage-dependent behavior as that induced by free H6 peptide added to the cis solution, thereby demonstrating that these residues remain on the cis side when T domain channels open. As expected, trans nickel had no effect on the gating of these channels.


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)

H6 peptide chemically linked at residues 293 or 320 blocks the T domain channel at positive voltages. Before the start of each record, T domain with H6 peptide attached at residue 293 (A) or 320 (B) was added to the cis compartment to a concentration of ∼300 (A) or 400 (B) ng/ml. (A) After addition of the T domain, conductance rose over time to the level seen at the start of the record, with the voltage held at +20 mV. When the voltage was switched to −20 mV, the conductance nearly doubled. Subsequent voltage steps to +30, +40, and +50 mV with intervening pulses to corresponding negative voltages demonstrated two facts. (a) The conductance at each positive test voltage was less than that at the corresponding negative voltage; the magnitude of the rectification increased with larger voltages. (b) Positive 10-mV increments produced disproportionately small current increases. (B) A single channel flickered between the open state and a zero-conductance blocked state at +60 mV, spending almost all its time in the zero-conductance state. When the voltage was switched to −60 mV, the channel became unblocked. Similar behavior was seen at ±80 mV after a 1-min break. (Records similar to those in A and B were obtained with T domain having the H6 peptide attached at residue 376, 291, or 294.) The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 30 mM Mes, pH 5.3, and the trans contained 50 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
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Figure 5: H6 peptide chemically linked at residues 293 or 320 blocks the T domain channel at positive voltages. Before the start of each record, T domain with H6 peptide attached at residue 293 (A) or 320 (B) was added to the cis compartment to a concentration of ∼300 (A) or 400 (B) ng/ml. (A) After addition of the T domain, conductance rose over time to the level seen at the start of the record, with the voltage held at +20 mV. When the voltage was switched to −20 mV, the conductance nearly doubled. Subsequent voltage steps to +30, +40, and +50 mV with intervening pulses to corresponding negative voltages demonstrated two facts. (a) The conductance at each positive test voltage was less than that at the corresponding negative voltage; the magnitude of the rectification increased with larger voltages. (b) Positive 10-mV increments produced disproportionately small current increases. (B) A single channel flickered between the open state and a zero-conductance blocked state at +60 mV, spending almost all its time in the zero-conductance state. When the voltage was switched to −60 mV, the channel became unblocked. Similar behavior was seen at ±80 mV after a 1-min break. (Records similar to those in A and B were obtained with T domain having the H6 peptide attached at residue 376, 291, or 294.) The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl2, 1 mM EDTA; the cis solution contained 30 mM Mes, pH 5.3, and the trans contained 50 mM HEPES, pH 7.2. The records were filtered at 100 Hz by the chart recorder.
Mentions: When the synthetic H6 peptide was tethered to either residue 291, 293, 294, 320, or 376, the T domain conductance declined very rapidly at positive voltages and increased very rapidly at negative voltages (Fig. 5 A). (The transients were too fast to be recorded at our 100-Hz filtering.) At the single-channel level, the channels spent most of their time in a zero-conductance blocked state at +60 or +80 mV; when the sign of the voltage was reversed, the channels recovered immediately to the open state (Fig. 5 B). Thus, at both the macroscopic and single-channel levels, channels with tethered H6 peptide at these residues qualitatively displayed the same voltage-dependent behavior as that induced by free H6 peptide added to the cis solution, thereby demonstrating that these residues remain on the cis side when T domain channels open. As expected, trans nickel had no effect on the gating of these channels.

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