Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin

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.     

Behavior of the deeply inserted helices in diphtheria toxin T domain: helices 5, 8, and 9 interact strongly and promote pore formation, while helices 6/7 limit pore formation

Diphtheria toxin T domain aids the membrane translocation of diphtheria toxin A chain. When the isolated T domain is deeply membrane-inserted, helices TH 8-9 form a transmembrane hairpin, while helices TH 5-7 form a deeply inserted nontransmembrane structure. Blocking deep insertion of TH 8-9 blocks deep insertion of TH 5-7 ( Zhao, G., and London, E. ( 2005) Biochemistry 44, 4488- 4498 ). We now examine the effects of blocking the deep insertion of TH 5 and TH 6/7. An A282R/V283R dual substitution in TH 5 prevented its deep insertion, significantly decreased the deep insertion of TH 9, and to a lesser degree that of TH 6/7. Blocking deep insertion of TH 6/7 with a L307R mutation had no effect on the deep insertion of TH 5, similar to its previously characterized lack of effect on the deep insertion of TH 8-9. An I364K mutation in TH 9 blocked TH 8-9 deep insertion and greatly reduced pore formation by the T domain, consistent with the role of TH 8-9 in pore formation. The A282R/V283R mutations also reduced the extent of pore formation, but to a lesser degree, suggesting either that TH 5 is part of the pore or that interactions with TH 5 affect the ability of TH 8-9 to form pores. The L307R mutation enhanced the extent of pore formation, suggesting that deeply inserted TH 6/7 may act as a "cork" that partly blocks the pore. Combined, these results indicate that TH 5, 8, and 9 combine to form a deeply inserted scaffold of more strongly associated helices.