Membrane translocation of charged residues at the tips of hydrophobic helices in the T domain of diphtheria toxin

The low pH triggered membrane insertion of the T domain of diphtheria toxin is a critical step in the translocation of the C domain of the toxin acros s membranes in vivo. We previously established that the T domain can intera ct with membranes in two distinct conformations, one in which the TH8/TH9 h elical hairpin lies close to the bilayer surface and a second in which it i nserts more deeply and appears to be transmembraneous. The loss of charge o n residues E349 and D352 due to protonation at low pH has been proposed to be a critical step in transmembrane insertion, because they are within a lo op connecting TH8 and TH9, and must cross the membrane upon transmembrane i nsertion. In this report, the role of these residues was examined by measur ing the effect of the double substitution E349K/D352K on the conformation o f the TH8/TH9 hairpin through a fluorescent group attached to TH9. At pH 4. 5, there was shallower insertion of TH8/TH9 of the E349K/D352K mutant relat ive to T domain with wild-type residues at 349 and 352. In addition, smalle r and/or fewer pores were obtained with the E349K/D352K mutant relative to the wild-type. On the other hand, high T domain concentrations, or further decreasing pH, allowed transmembrane insertion of both the wild-type and th e 349K/352K mutant as well as induction of larger and/or more numerous pore s. Furthermore, the transmembrane insertion process was rapid fur both the mutant and wild-type. This shows that the mutant has the capacity to form a transmembrane structure similar to that of the wild-type T domain and, thu s, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane move ment, The linkage between the ability of the T domain to form the transmemb rane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability t o insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues oth er than E349 and D352 whose protonation promotes transmembrane insertion.