The ␣-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes. In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane. (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer.) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids. It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface. However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane. In contrast, residue 322 remains exposed to aqueous solution under these conditions. These conclusions were confirmed by a novel antibody binding method. Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene (BODIPY) groups were used to evaluate the exposure of BODIPYlabeled 322, 333, and 356. Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer. In contrast, residue 322 remained exposed under all conditions. We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state. The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation. Our results suggest that toxintoxin interactions may play an important role in regulating this behavior.Diphtheria toxin is a protein secreted by Corynebacterium diphtheriae. It can be split into two chains, A (21 kDa) and B (37 kDa), joined by a disulfide bond (1). The crystal structure of the toxin shows that it consists of three domains (2-5). The A chain is the catalytic (C) domain. The B chain contains the transmembrane (T) and receptor binding (R) domains. Membrane penetration is believed to occur after the toxin reaches endosomes (6). The low pH within the endosomal lumen induces a partial unfolding of the toxin, resulting in exposure of the hydrophobic regions and translocation of the A chain of the toxin into the cytoplasm (6). Once in the cytoplasm, the A chain catalyzes the transfer of the ADP-ribosyl group of NAD ϩ to elongation factor 2, inactivating protein synthesis.The T domain is made up of nine ␣-helices, several of which contain hydrophobic sequences that play a critical role in...

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.Diphtheria toxin is secreted by Corynebacterium diphtheriae. The toxin can be split into two chains, A (M r 21 kDa) and B (M r 37 kDa), joined by a disulfide bond (1). The crystal structure shows diphtheria toxin consists of three domains (2-5). The A chain is the catalytic (C) domain. The B chain contains the transmembrane (T) and receptor-binding (R) domains. Subsequent to endocytosis, the toxin reaches endosomes and membrane penetration occurs (6). The low pH within the endosomal lumen induces a partial unfolding of the toxin, resulting in exposure of the hydrophobic regions and translocation of the A chain of the toxin into the cytoplasm (6). In the cytoplasm, the A chain catalyzes the transfer of ADP-ribosyl group of NAD ϩ to elongation factor 2, inactivating protein synthesis.The T domain is composed basically of ␣-helices, several of which are hydrophobic and are likely to play a critical role in membrane insertion and translocation (2, 7). Determining whether such helical hydrophobic segments span the bilayer has been an especially difficult problem for those proteins that, like the T domain, convert between soluble and membraneinserted forms. For example, despite much study the boundaries of the inserted sequence and the orientation of the hydrophobic tail of cytochrome b 5 in its loosely and tightly bound states has proven complex (8, 9)...

The transmembrane (T) domain of diphtheria toxin has a critical role in the low pH-induced translocation of the catalytic domain (A chain) of the toxin across membranes. Here it is shown that at low pH, addition of proteins in a partly unfolded, molten globule-like conformation converted the T domain from a shallow membrane-inserted form to its transmembrane form. Fluorescence energy transfer demonstrated that molten globule-like proteins bound to the T domain. Thus, the T domain recognizes proteins that are partly unfolded and may function in translocation of the A chain as a transmembrane chaperone.

The role of Trp and Tyr residues in determining membrane protein structure is particularly interesting because indole and phenol structures combine hydrophobic and polar groups, and it is hard to predict the exact region of the membrane at which their energy would be at a minimum. To determine the depths intrinsically favored by these residues, the locations of membrane-associating Trp and Tyr analogs have been determined using a fluorescence quenching technique able to measure depth at high resolution. They are found to locate at the same depths as Trp and Tyr in membrane proteins, 14-15 A from the bilayer center, which implies an important role for these residues in aligning membrane proteins in precise relationship to the lipid bilayer.