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

Paul K. Kienker, Zhengyan Wu, Alan Finkelstein

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)107-125
Number of pages19
JournalJournal of General Physiology
Volume145
Issue number2
DOIs
StatePublished - 2015

Fingerprint

Diphtheria Toxin
Cysteine
His-His-His-His-His-His
Membranes
Lipid Bilayers
Constriction
Sulfhydryl Reagents
Catalytic Domain

ASJC Scopus subject areas

  • Physiology

Cite this

Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel. / Kienker, Paul K.; Wu, Zhengyan; Finkelstein, Alan.

In: Journal of General Physiology, Vol. 145, No. 2, 2015, p. 107-125.

Research output: Contribution to journalArticle

Kienker, Paul K. ; Wu, Zhengyan ; Finkelstein, Alan. / Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel. In: Journal of General Physiology. 2015 ; Vol. 145, No. 2. pp. 107-125.
@article{9bf5af60be794ade8777ca8fcc7a79b7,
title = "Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel",
abstract = "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.",
author = "Kienker, {Paul K.} and Zhengyan Wu and Alan Finkelstein",
year = "2015",
doi = "10.1085/jgp.201411326",
language = "English (US)",
volume = "145",
pages = "107--125",
journal = "Journal of General Physiology",
issn = "0022-1295",
publisher = "Rockefeller University Press",
number = "2",

}

TY - JOUR

T1 - Mapping the membrane topography of the TH6-TH7 segment of the diphtheria toxin T-domain channel

AU - Kienker, Paul K.

AU - Wu, Zhengyan

AU - Finkelstein, Alan

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84921790392&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84921790392&partnerID=8YFLogxK

U2 - 10.1085/jgp.201411326

DO - 10.1085/jgp.201411326

M3 - Article

C2 - 25582482

AN - SCOPUS:84921790392

VL - 145

SP - 107

EP - 125

JO - Journal of General Physiology

JF - Journal of General Physiology

SN - 0022-1295

IS - 2

ER -