{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T01:12:31Z","timestamp":1790817151246,"version":"4.1.0"},"reference-count":27,"publisher":"Rockefeller University Press","issue":"4","content-domain":{"domain":["rupress.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2000,4,1]]},"abstract":"<jats:p>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 \u201cdouble dagger\u201d 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 \u201cdaggers,\u201d 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 \u223c70 residues, is translocated across the membrane to the trans side.<\/jats:p>","DOI":"10.1085\/jgp.115.4.421","type":"journal-article","created":{"date-parts":[[2002,7,26]],"date-time":"2002-07-26T16:50:20Z","timestamp":1027702220000},"page":"421-434","update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":69,"title":["Topography of Diphtheria Toxin's T Domain in the Open Channel State"],"prefix":"10.1085","volume":"115","author":[{"given":"Lisa","family":"Senzel","sequence":"first","affiliation":[{"name":"aFrom the Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Gordon","sequence":"additional","affiliation":[{"name":"aFrom the Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert O.","family":"Blaustein","sequence":"additional","affiliation":[{"name":"cDepartment of Biochemistry, Brandeis University, Waltham, Massachusetts 02254"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"K. Joon","family":"Oh","sequence":"additional","affiliation":[{"name":"dDepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"R. John","family":"Collier","sequence":"additional","affiliation":[{"name":"dDepartment of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alan","family":"Finkelstein","sequence":"additional","affiliation":[{"name":"aFrom the Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461"},{"name":"bFrom the Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"291","published-online":{"date-parts":[[2000,4,1]]},"reference":[{"key":"2023072616093513900_Bennettetal1994","doi-asserted-by":"crossref","first-page":"1444","DOI":"10.1002\/pro.5560030911","article-title":"Refined structure of dimeric diphtheria toxin at 2.0 \u00c5 resolution","volume":"3","author":"Bennett","year":"1994","journal-title":"Prot. Sci."},{"key":"2023072616093513900_Choeetal1992","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1038\/357216a0","article-title":"The crystal structure of diphtheria toxin","volume":"357","author":"Choe","year":"1992","journal-title":"Nature."},{"key":"2023072616093513900_Donovanetal1981","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1073\/pnas.78.1.172","article-title":"Diphtheria toxin forms transmembrane channels in planar lipid bilayers","volume":"78","author":"Donovan","year":"1981","journal-title":"Proc. Natl. Acad. Sci. USA."},{"key":"2023072616093513900_DraperandSimon1980","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1083\/jcb.87.3.849","article-title":"The entry of diphtheria toxin into the mammalian cell cytoplasmevidence for lysosomal involvement","volume":"87","author":"Draper","year":"1980","journal-title":"J.Cell Biol."},{"key":"2023072616093513900_Eriksenetal1994","doi-asserted-by":"crossref","first-page":"4433","DOI":"10.1002\/j.1460-2075.1994.tb06765.x","article-title":"Diphtheria toxin at low pH depolarizes the membrane, increases the membrane conductance and induces a new type of ion channel in Vero cells","volume":"13","author":"Eriksen","year":"1994","journal-title":"EMBO (Eur. Mol. Biol. Organ.) J."},{"key":"2023072616093513900_Falnesetal1992","doi-asserted-by":"crossref","first-page":"12284","DOI":"10.1016\/S0021-9258(19)49836-0","article-title":"Replacement of negative by positive charges in the presumed membrane-inserted part of diphtheria toxin B fragmenteffect on membrane translocation and on formation of channels","volume":"267","author":"Falnes","year":"1992","journal-title":"J. Biol. Chem."},{"key":"2023072616093513900_Greenfieldetal1983","doi-asserted-by":"crossref","first-page":"6853","DOI":"10.1073\/pnas.80.22.6853","article-title":"Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage b","volume":"80","author":"Greenfield","year":"1983","journal-title":"Proc. Natl. Acad. Sci. USA."},{"key":"2023072616093513900_Huynhetal1997","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1085\/jgp.110.3.229","article-title":"Probing the structure of the diphtheria toxin channelreactivity in planar lipid bilayer membranes of cysteine-substituted mutant channels with methanethiosulfonate derivatives","volume":"110","author":"Huynh","year":"1997","journal-title":"J. Gen. Physiol."},{"key":"2023072616093513900_Kacheletal1998","doi-asserted-by":"crossref","first-page":"22950","DOI":"10.1074\/jbc.273.36.22950","article-title":"Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain","volume":"273","author":"Kachel","year":"1998","journal-title":"J. Biol. Chem."},{"key":"2023072616093513900_Kaganetal1981","doi-asserted-by":"crossref","first-page":"4950","DOI":"10.1073\/pnas.78.8.4950","article-title":"Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes","volume":"78","author":"Kagan","year":"1981","journal-title":"Proc. Natl. Acad. Sci. USA."},{"key":"2023072616093513900_Lanzreinetal1997","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/s002329900196","article-title":"Structure\u2013function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes","volume":"156","author":"Lanzrein","year":"1997","journal-title":"J. Membr. Biol."},{"key":"2023072616093513900_MadshusandStenmark1992","first-page":"1","article-title":"Entry of ADP-ribosylating toxins into cells","volume":"175","author":"Madshus","year":"1992","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"2023072616093513900_Mindelletal1994a","first-page":"29","article-title":"Structure\u2013function relationships in diphtheria toxin channels. II. A residue responsible for the channel's dependence on trans pH","volume":"137","author":"Mindell","year":"1994","journal-title":"J. Membr. Biol."},{"key":"2023072616093513900_Mindelletal1994b","first-page":"45","article-title":"Structure\u2013function relationships in diphtheria toxin channelsIII. Residues which affect the cis pH dependence of channel conductance","volume":"137","author":"Mindell","year":"1994","journal-title":"J. Membr. Biol."},{"key":"2023072616093513900_Moskaugetal1991","doi-asserted-by":"crossref","first-page":"2652","DOI":"10.1016\/S0021-9258(18)52294-8","article-title":"Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH","volume":"266","author":"Moskaug","year":"1991","journal-title":"J. Biol. Chem."},{"key":"2023072616093513900_NeilsenandBuchardt1991","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1055\/s-1991-26579","article-title":"Facile synthesis of reagents containing a terminal maleimido ligand linked to an active ester","volume":"10","author":"Neilsen","year":"1991","journal-title":"Synthesis."},{"key":"2023072616093513900_Ohetal1999","doi-asserted-by":"crossref","first-page":"8467","DOI":"10.1073\/pnas.96.15.8467","article-title":"Translocation of the catalytic domain of diphtheria toxin across planar phospholipid bilayers by its own T domain","volume":"96","author":"Oh","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA."},{"key":"2023072616093513900_Ohetal1996","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1126\/science.273.5276.810","article-title":"Organization of diphtheria toxin T domain in bilayersa site-directed spin labeling study","volume":"273","author":"Oh","year":"1996","journal-title":"Science."},{"key":"2023072616093513900_Qiuetal1994","doi-asserted-by":"crossref","first-page":"7483","DOI":"10.1016\/S0021-9258(17)37311-8","article-title":"Site-specific biotinylation of colicin Iaa probe for protein conformation in the membrane","volume":"269","author":"Qiu","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2023072616093513900_Qiuetal1996","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1085\/jgp.107.3.313","article-title":"Major transmembrane movement associated with colicin Ia channel gating","volume":"107","author":"Qiu","year":"1996","journal-title":"J. Gen. Physiol."},{"key":"2023072616093513900_Quertenmontetal1996","doi-asserted-by":"crossref","first-page":"1283","DOI":"10.1046\/j.1365-2958.1996.851446.x","article-title":"Topology of diphtheria toxin in lipid vesicle membranesa proteolysis study","volume":"21","author":"Quertenmont","year":"1996","journal-title":"Mol. Microbiol."},{"key":"2023072616093513900_SandvigandOlsnes1980","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1083\/jcb.87.3.828","article-title":"Diphtheria toxin entry into cells is facilitated by low pH","volume":"87","author":"Sandvig","year":"1980","journal-title":"J. Cell Biol."},{"key":"2023072616093513900_Senzeletal1998","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1085\/jgp.112.3.317","article-title":"The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region across planar bilayers","volume":"112","author":"Senzel","year":"1998","journal-title":"J. Gen. Physiol."},{"key":"2023072616093513900_Silvermanetal1994a","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1007\/BF00234995","article-title":"Structure\u2013function relationships in diphtheria toxin channelsdetermining a minimal channel-forming domain","volume":"137","author":"Silverman","year":"1994","journal-title":"J. Membr. Biol."},{"key":"2023072616093513900_Silvermanetal1994b","doi-asserted-by":"crossref","first-page":"22524","DOI":"10.1016\/S0021-9258(17)31678-2","article-title":"Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain","volume":"269","author":"Silverman","year":"1994","journal-title":"J. Biol. Chem."},{"key":"2023072616093513900_Wangetal1997","doi-asserted-by":"crossref","first-page":"16300","DOI":"10.1021\/bi971281z","article-title":"Use of Trp mutations to evaluate the conformational behavior and membrane insertion of A and B chains in whole diphtheria toxin","volume":"36","author":"Wang","year":"1997","journal-title":"Biochemistry."},{"key":"2023072616093513900_Zhanetal1995","doi-asserted-by":"crossref","first-page":"4856","DOI":"10.1021\/bi00014a043","article-title":"Interaction of the isolated transmembrane domain of diphtheria toxin with membranes","volume":"34","author":"Zhan","year":"1995","journal-title":"Biochemistry."}],"container-title":["The Journal of General Physiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/rupress.org\/jgp\/article-pdf\/115\/4\/421\/1774547\/gp-8148.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/rupress.org\/jgp\/article-pdf\/115\/4\/421\/1774547\/gp-8148.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,7,26]],"date-time":"2023-07-26T19:17:52Z","timestamp":1690399072000},"score":1,"resource":{"primary":{"URL":"https:\/\/rupress.org\/jgp\/article\/115\/4\/421\/51216\/Topography-of-Diphtheria-Toxin-s-T-Domain-in-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,4,1]]},"references-count":27,"journal-issue":{"issue":"4","published-print":{"date-parts":[[2000,4,1]]}},"URL":"https:\/\/doi.org\/10.1085\/jgp.115.4.421","relation":{},"ISSN":["0022-1295","1540-7748"],"issn-type":[{"value":"0022-1295","type":"print"},{"value":"1540-7748","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,4,1]]}}}