title: Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel.
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.
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA. kienker@outlook.com.
authors.forename: Paul K
authors.initials: PK
authors.lastname: Kienker
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.|Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, Anhui, China.
authors.forename: Zhengyan
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.|Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Finkelstein
mesh_terms: D000465:Algorithms; D003545:Cysteine / Q000737:chemistry; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D054730:Protein Interaction Domains and Motifs / Q000235:genetics
chemical_list: D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D003545:Cysteine
keywords: Bacterial toxins; Ion-conducting channels; Kinetics; Lipid bilayers; Sulfhydryl reagents