scholarly journals Topography of Diphtheria Toxin's T Domain in the Open Channel State

2000 ◽  
Vol 115 (4) ◽  
pp. 421-434 ◽  
Author(s):  
Lisa Senzel ◽  
Michael Gordon ◽  
Robert O. Blaustein ◽  
K. Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Catalytic Domain ◽  
Water Soluble ◽  
Soluble Form ◽  
Crystallographic Structure ◽  
Channel State ◽  
Amino Terminal ◽  
T Domain ◽  
Membrane Spanning

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.

scholarly journals Evidence for a TH6-TH7 Transmembrane Hairpin in the Diphtheria Toxin T-Domain Open Channel State

2012 ◽  
Vol 102 (3) ◽  
pp. 657a
Author(s):  
Paul Kienker ◽  
Zhengyan Wu ◽  
Alan Finkelstein
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Channel State ◽  
T Domain

scholarly journals Crucial Role of H322 in the Folding of Diphtheria Toxin T-Domain into the Open-Channel State

2014 ◽  
Vol 106 (2) ◽  
pp. 717a
Author(s):  
Mauricio Vargas-Uribe ◽  
Mykola V. Rodnin ◽  
Paul Kienker ◽  
Alan Finkelstein ◽  
Alexey S. Ladokhin
Keyword(s):  
Crucial Role ◽  
Diphtheria Toxin ◽  
Open Channel ◽  
Channel State ◽  
T Domain

scholarly journals Crucial Role of H322 in Folding of the Diphtheria Toxin T-Domain into the Open-Channel State

Biochemistry ◽  
2013 ◽  
Vol 52 (20) ◽  
pp. 3457-3463 ◽  
Author(s):  
Mauricio Vargas-Uribe ◽  
Mykola V. Rodnin ◽  
Paul Kienker ◽  
Alan Finkelstein ◽  
Alexey S. Ladokhin
Keyword(s):  
Crucial Role ◽  
Diphtheria Toxin ◽  
Open Channel ◽  
Channel State ◽  
T Domain

scholarly journals Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

2015 ◽  
Vol 145 (2) ◽  
pp. 107-125 ◽  
Author(s):  
Paul K. Kienker ◽  
Zhengyan Wu ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Single Channel ◽  
Reaction Rates ◽  
Cysteine Residue ◽  
Channel Conductance ◽  
Amino Terminal ◽  
Substituted Cysteine Accessibility ◽  
T Domain

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.

scholarly journals Probing the Structure of the Diphtheria Toxin Channel

1997 ◽  
Vol 110 (3) ◽  
pp. 229-242 ◽  
Author(s):  
Paul D. Huynh ◽  
Can Cui ◽  
Hangjun Zhan ◽  
Kyoung Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Single Channel ◽  
Sudden Change ◽  
Water Soluble ◽  
Soluble Form ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Conducting Pathway ◽  
Ion Conducting ◽  
T Domain

Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328–376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α-helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.

scholarly journals Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa

2005 ◽  
Vol 385 (3) ◽  
pp. 667-675 ◽  
Author(s):  
Susan P. YATES ◽  
Patricia L. TAYLOR ◽  
René JØRGENSEN ◽  
Dana FERRARIS ◽  
Jie ZHANG ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Competitive Inhibition ◽  
Function Analysis ◽  
Binding Pocket ◽  
Ring Structure ◽  
Water Soluble ◽  
Exotoxin A ◽  
Ic50 Values

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant Ki of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å (1 Å=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

scholarly journals Cellular Entry of the Diphtheria Toxin Does Not Require the Formation of the Open-Channel State by Its Translocation Domain

Toxins ◽  
2017 ◽  
Vol 9 (10) ◽  
pp. 299 ◽  
Author(s):  
Alexey Ladokhin ◽  
Mauricio Vargas-Uribe ◽  
Mykola Rodnin ◽  
Chiranjib Ghatak ◽  
Onkar Sharma
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Channel State ◽  
Translocation Domain ◽  
Cellular Entry

scholarly journals The Number of Subunits Comprising the Channel Formed by the T Domain of Diphtheria Toxin

2001 ◽  
Vol 118 (5) ◽  
pp. 471-480 ◽  
Author(s):  
Michael Gordon ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Low Ph ◽  
Voltage Gating ◽  
Single Channels ◽  
Planar Lipid Bilayers ◽  
Transmembrane Segments ◽  
T Domain ◽  
Single Subunit

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine+ and NAD−, it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH2-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.

scholarly journals Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain

2000 ◽  
Vol 116 (4) ◽  
pp. 587-598 ◽  
Author(s):  
Paul K. Kienker ◽  
Karen S. Jakes ◽  
Alan Finkelstein
Keyword(s):  
Protein Translocation ◽  
Bilayer Membrane ◽  
Water Soluble ◽  
Planar Bilayers ◽  
Amino Terminal ◽  
Water Soluble Protein ◽  
Voltage Gated Channels ◽  
Membrane Spanning ◽  
Carboxy Terminal ◽  
Colicin Ia

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an ∼68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.

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