Organization of diphtheria toxin in membranes - A hydrophobic photolabeling study

Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a cataly tic domain (C), a membrane-inserting domain (T), and a receptor-binding dom ain (R). It gains entry into cells by receptor-mediated endocytosis. The lo w pH (similar to 5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subseque nt translocation of the C domain into the cell, where it inactivates protei n synthesis ultimately leading to cell death. We have used a highly reactiv e hydrophobic photoactivable reagent, DAF, to identify the segments of DT t hat interact with the membrane at pH 5.2. This reagent readily partitions i nto 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 he lices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfi de link was found on the trans side leaving part of the C domain on the tra ns side. This domain then comes out to the cis side via a highly hydrophobi c patch corresponding to residues 134-141, originally corresponding to a be ta-strand in the solution structure of DT. It appears that the three helice s 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 dis ulfide reductase separates the two chains.