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Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel

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Abstract

The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1–TH4 translocated across the membrane. The TH6–TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.

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Abbreviations

C-domain:

Catalytic domain of DT

DT:

Diphtheria toxin

DTT:

Dithiothreitol

His6-tag:

Hexahistidine tag

I–V :

Current–voltage

MTS:

Methanethiosulfonate

MTS-ACE:

[2-(Aminocarbonyl)ethyl] MTS

MTS-ET:

[2-(Trimethylammonium)ethyl] MTS bromide

SCAM:

Substituted-cysteine accessibility method

T-domain:

Translocation domain of DT

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Acknowledgments

This work was supported by the National Institutes of Health Grant GM29210.

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Author notes

  1. Zhengyan Wu

    Present address: Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, China

Authors and Affiliations

  1. Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Paul K. Kienker, Zhengyan Wu & Alan Finkelstein

  2. Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA

    Alan Finkelstein

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  1. Paul K. Kienker
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Correspondence to Paul K. Kienker.

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Kienker, P.K., Wu, Z. & Finkelstein, A. Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel. J Membrane Biol 249, 181–196 (2016). https://doi.org/10.1007/s00232-015-9859-9

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