The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.
Diphtheria toxin (DT)1 is a protein toxin secreted by the bacterium Corynebacterium diphtheriae. The mature toxin (58 kDa) consists of two polypeptide chains, A (21 kDa) and B (37 kDa), which are joined by a single disulfide bond. Solution of the crystal structure at neutral pH revealed that the toxin consists of three distinct domains. The A chain is identical to the catalytic (C) domain of the protein while the B chain consists of two domains, the receptor-binding domain (R) and the transmembrane (T) domain (2-5). Upon entering endosomes, via receptor-mediated endocytosis, a confomational change triggered by the low pH of the endosomal lumen renders the toxin hydrophobic. This results in membrane penetration by the toxin and subsequent translocation of the A chain into the cytosol. Once in the cytosol, the A chain catalyzes the ADPribosylation of the diphthamide residue of elongation factor 2, which shuts down protein synthesis and leads to cell death (6).The details of the mechanisms by which protein toxins such as DT (or for that matter any proteins) translocate across membranes remain elusive. One reason is that toxins with helical membrane-inserted segments are challenging targets for topography studi...