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 across membranes in vivo. We previously established that the T domain can interact with membranes in two distinct conformations, one in which the TH8/TH9 helical hairpin lies close to the bilayer surface and a second in which it inserts more deeply and appears to be transmembraneous. The loss of charge on 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 loop connecting TH8 and TH9, and must cross the membrane upon transmembrane insertion. In this report, the role of these residues was examined by measuring the effect of the double substitution E349K/D352K on the conformation of 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 relative to T domain with wild-type residues at 349 and 352. In addition, smaller 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 the 349K/352K mutant as well as induction of larger and/or more numerous pores. 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, thus, that introduction of charged groups in membrane-penetrating regions of a protein does not introduce an insurmountable barrier to transmembrane movement, The linkage between the ability of the T domain to form the transmembrane conformation and pores suggests that the effects of these mutations in inhibiting pore formation are likely to partly result from the inability to insert properly. Additionally, the observation that decreasing pH allows the 349K/352K mutant to insert deeply indicates that there are residues other than E349 and D352 whose protonation promotes transmembrane insertion.