title: Organization of diphtheria toxin in membranes. A hydrophobic photolabeling study.
abstract: Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis. The low pH ( approximately 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134-141, originally corresponding to a beta-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.
authors.affiliation: Biomembrane Laboratory, Department of Chemistry and Biotechnology Center, Indian Institute of Technology Bombay, Powai, Bombay 400 076, India.
authors.forename: P R
authors.initials: PR
authors.lastname: D'Silva
authors.forename: A K
authors.initials: AK
authors.lastname: Lala
mesh_terms: D000345:Affinity Labels; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D003979:Diazonium Compounds; D004167:Diphtheria Toxin / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005449:Fluorenes; D008567:Membranes, Artificial; D008958:Models, Molecular; D010449:Peptide Mapping; D010782:Photolysis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D003979:Diazonium Compounds; D004167:Diphtheria Toxin; D004220:Disulfides; D005449:Fluorenes; D008567:Membranes, Artificial; C052770:2-diazofluorene