ROLES OF GLU-349 AND ASP-352 IN MEMBRANE INSERTION AND TRANSLOCATION BY DIPHTHERIA-TOXIN

Acidic conditions within the endosomal lumen induce the T domain of re ceptor-bound diphtheria toxin (DT) to insert into the endosomal membra ne and mediate translocation of the toxin's catalytic domain to the cy tosol. A conformational rearrangement in the toxin occurring near pH 5 allows a buried apolar helical hairpin of the native T domain (helice s TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues withi n the TL5 interhelical loop, Glu 349 and Asp 352, should be come expos ed at the neutral cytosolic face of the membrane and reionize. To inve stigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced w ith nonionizable ones. Each of two double mutants examined showed a se veral-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A+D352A and fourfold for E349Q+D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to per meabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double muta tions affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demon strate that, although there is no absolute requirement for an acidic r esidue within the TL5 loop for toxicity, Glu 349 and Asp 352 do signif icantly enhance the biological activity of the protein. The data are c onsistent with a model in which ionization of these residues at the cy tosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formati on and translocation of the toxin's catalytic chain.