Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state

Mauricio Vargas-Uribe, Mykola V. Rodnin, Paul Kienker, Alan Finkelstein, Alexey S. Ladokhin

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

Original languageEnglish (US)
Pages (from-to)3457-3463
Number of pages7
JournalBiochemistry
Volume52
Issue number20
DOIs
StatePublished - May 21 2013

Fingerprint

Diphtheria Toxin
Membranes
Acidification
Histidine
Regain
Lipid bilayers
Lipid Bilayers
Circular Dichroism
Glutamine
Tryptophan
Assays
Substitution reactions
Fluorescence
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Vargas-Uribe, M., Rodnin, M. V., Kienker, P., Finkelstein, A., & Ladokhin, A. S. (2013). Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. Biochemistry, 52(20), 3457-3463. https://doi.org/10.1021/bi400249f

Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. / Vargas-Uribe, Mauricio; Rodnin, Mykola V.; Kienker, Paul; Finkelstein, Alan; Ladokhin, Alexey S.

In: Biochemistry, Vol. 52, No. 20, 21.05.2013, p. 3457-3463.

Research output: Contribution to journalArticle

Vargas-Uribe, M, Rodnin, MV, Kienker, P, Finkelstein, A & Ladokhin, AS 2013, 'Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state', Biochemistry, vol. 52, no. 20, pp. 3457-3463. https://doi.org/10.1021/bi400249f
Vargas-Uribe M, Rodnin MV, Kienker P, Finkelstein A, Ladokhin AS. Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. Biochemistry. 2013 May 21;52(20):3457-3463. https://doi.org/10.1021/bi400249f
Vargas-Uribe, Mauricio ; Rodnin, Mykola V. ; Kienker, Paul ; Finkelstein, Alan ; Ladokhin, Alexey S. / Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state. In: Biochemistry. 2013 ; Vol. 52, No. 20. pp. 3457-3463.
@article{f716c1d6d1854e59bb1c74eacdc9a8f7,
title = "Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state",
abstract = "The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.",
author = "Mauricio Vargas-Uribe and Rodnin, {Mykola V.} and Paul Kienker and Alan Finkelstein and Ladokhin, {Alexey S.}",
year = "2013",
month = "5",
day = "21",
doi = "10.1021/bi400249f",
language = "English (US)",
volume = "52",
pages = "3457--3463",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "20",

}

TY - JOUR

T1 - Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state

AU - Vargas-Uribe, Mauricio

AU - Rodnin, Mykola V.

AU - Kienker, Paul

AU - Finkelstein, Alan

AU - Ladokhin, Alexey S.

PY - 2013/5/21

Y1 - 2013/5/21

N2 - The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

AB - The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

UR - http://www.scopus.com/inward/record.url?scp=84878229869&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84878229869&partnerID=8YFLogxK

U2 - 10.1021/bi400249f

DO - 10.1021/bi400249f

M3 - Article

C2 - 23621842

AN - SCOPUS:84878229869

VL - 52

SP - 3457

EP - 3463

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 20

ER -