Membrane topography of the T domain of diphtheria toxin probed with singletryptophan mutants

The membrane insertion and translocation of diphtheria toxin, which is indu ced in vivo by low pH, is thought to be directed by the hydrophobic a-helic es of its transmembrane (T) domain. In this study the structure of membrane -associated T domain was examined. Site-directed mutants of the T domain wi th single Trp residues were prepared at the two naturally occurring positio ns, 206 (near the N-terminal end of helix TH1) and 281 (within helix TH5), as well as at three residues in helix TH9, in which the substitutions F355W (near the N-terminal end of TH9), I364W (close to the center of TH9), and Y375W (near the C-terminal end of TH9) were made. All these mutants were fo und to undergo the low-pH induced conformational change observed with wild- type T domain and insert into model membranes at low pH. The location of Tr p residues relative to the lipid bilayer was characterized in model membran e vesicles by fluorescence emission and by quenching with nitroxide-labeled phospholipids. In TH9, residue 375 was shallowly inserted, residue 364 dee ply inserted, and residue 355 located at an intermediate depth. Residues 20 6 and 281 exhibited moderately deep insertion. It was also found, in agreem ent with our previous study using bimane-labeled protein (Wang et al. (1997 ) J. Biol. Chem. 272, 25091-25098), that TH9 switches from a relatively sha llowly inserted state to a more deeply inserted state when the concentratio n of the T domain in the membrane is increased or the thickness of the memb rane bilayer is decreased. In particular, the depth of residue 355 was foun d to increase under the conditions giving deeper insertion. In contrast, re sidue 375 remained shallowly located in both states, as predicted from its location on the polar C-terminus of TH9. It is concluded that TH1 and TH5 i nsert into the lipid bilayer in both T domain conformations. In addition, T rp depths suggest that even in the shallowly inserted state there is a sign ificant degree of insertion of TH9. These results suggest regions of the T domain in addition to the hydrophobic TH8/TH9 hairpin insert into membranes . Models for the structure of the membrane-inserted T domain are discussed.