Fig. 6. AT and PT have functional translocases with altered pH dependence.
a Overlay of T-domain structures (AT: 20% sequence identity, RMSD 3.0 Å; PT: 31% sequence identity, RMSD 3.1 Å). b Residues of interest in the TH8/TH9 loop involved in initial membrane insertion of DTT (top). AT and PT contain an equivalent to DTP345 (ATP369 and PTP386). The loop in AT contains a three-residue insertion and an additional Pro residue within the loop (ATP373), and only one negatively charged residue (ATD379). PTP386 causes a 90° kink in TH8, which continues for a full turn before terminating with a Glu residue (PTE390). Residues of interest at the junction of TH1, TH2, and TH3 believed to play an important role in early destabilization of DTT (bottom). ATT contains two salt-bridges (D252/R282, D257/K278) in lieu of His residues in this junction. c Toxicity of chimeric toxins on Vero cells. Dose titration of DT and DT T-domain chimeras DT(ATT), and DT(PTT) on Vero cells (mean ± SD, n = 2); n = 3 biological replicates. d pH dependent TNS analysis (mean ± SD, n = 3). e pH-dependent dye leakage from liposomes loaded with fluorophore quencher pair (HPTS/DPX). DT exhibits a spike of activity upon exposure to pH 5, while AT and PT are only active at pH 4, a full pH unit lower than DT (mean ± SD, n = 4); n = 3 biological replicates.