Conformation of the diphtheria toxin T domain in membranes: A site-directed spin-labeling study of the TH8 helix and TL5 loop

The isolated T domain of diphtheria toxin was mutated by cysteine-scanning mutagenesis at 28 consecutive sites (residues 328-355) that comprise the TH 8 helix and the TL5 interhelical loop in the native toxin. After derivatizi ng the mutant proteins with a sulfhydryl-selective nitroxide reagent, we ex amined the mobility of each nitroxide and its accessibility to polar and no npolar paramagnetic reagents, before and after insertion into phospholipid bilayers. The data obtained with the proteins in solution at pH 8 are gener ally consistent with predictions from the crystal structure of the toxin. U pon membrane binding at pH 4.6, a major structural reorganization of the do main was seen, which dramatically reduced the accessibility of most residue s in this region to the polar reagent nickel(II)-ethylenediaminediacetate c omplex (NiEDDA). Many of these residues also showed reduced accessibility t o the nonpolar reagent O-2. Periodic accessibility of the nitroxide side ch ains along the sequence to these reagents shows that TH8 remains largely he lical in the membrane-bound state, with one surface associated with protein and the other facing the hydrophobic interior of the bilayer. In addition, the TL5 loop also appears to become alpha-helical in the membrane, with on e surface in contact with protein and the other in contact with the bilayer interior. These findings provide a structural framework for understanding how the T domain forms a transmembrane channel and mediates translocation o f diphtheria toxin's enzymic moiety across a membrane.