MAINTENANCE OF THE HYDROPHOBIC FACE OF THE DIPHTHERIA-TOXIN AMPHIPATHIC TRANSMEMBRANE HELIX-1 IS ESSENTIAL FOR THE EFFICIENT DELIVERY OF THE CATALYTIC DOMAIN TO THE CYTOSOL OF TARGET-CELLS

The transmembrane (T) domain of diphtheria toxin (DT) comprises nine a lpha-helices and has been shown to play an essential role in the effic ient delivery of the catalytic (C) domain of DT across the eukaryotic cell membrane and into the cytosol. We have demonstrated recently that the first three amphipathic helixes of the T domain, although not nec essary for either channel formation or receptor binding, are required for the efficient transmembrane delivery of the C domain. In the prese nt study, we have performed a detailed structure - function analysis o f T domain helix 1 (TH1) of the DT -related fusion protein DAB(389)IL- 2. We performed exchange and site-directed mutagenesis of TH1 and the resulting mutant fusion toxins were analyzed by gel electrophoresis an d tested for their efficiencies in the delivery of the C domain to the cell cytosol. We demonstrate that the overall charge distribution and hydrophobicity of amino acids in the amphipathic helix TH1, rather th an a specific amino acid sequence, are critical for the function of th is helix. The insertion of a charged residue in the hydrophobic face o f TH1 abolishes cytotoxic activity, whereas replacement of a hydrophob ic residue by a charged amino acid in the hydrophilic face of the heli x has little, if any, effect on cytotoxic activity. In addition, we ha ve identified Ser220 by site-directed mutagenesis as a residue that ap pears to be critical for correct folding of the fusion toxin. Mutation s in this position result in fusion proteins that are extremely sensit ive to proteolytic attack.