Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes

Translocation of the catalytic domain of diphtheria toxin (DT) across the e ndosomal membrane to the cytoplasm of mammalian cells requires the low-pH-d ependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is burie d within the T domain of the native protein. Mutations of Pro345, which ter minates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phosp holipid bilayers and in permeabilizing phospholipid vesicles to entrapped f luorophores. Experiments with isolated T domain containing a polarity-sensi tive fluorophore attached to Cys at position 332 suggest that the P345E mut ation blocks membrane insertion. None of the Pro345 mutations shifted the p H-dependence of binding in solution of the hydrophobic fluorophore, 2-p-tol uidinyl-naphthalene 7-sulfonate. The results indicate that proline at posit ion 345 is required for the T domain to insert into phospholipid bilayers o r to adopt a functional conformation within the bilayer.