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 alpha-helices and has been shown to play an essential role in the efficient 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 necessary for either channel formation or receptor binding, are required for the efficient transmembrane delivery of the C domain. In the present study, we have performed a detailed structure-function analysis of T domain helix 1 (TH1) of the DT-related fusion protein DAB389IL-2. We performed exchange and site-directed mutagenesis of TH1 and the resulting mutant fusion toxins were analyzed by gel electrophoresis and 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 than a specific amino acid sequence, are critical for the function of this helix. The insertion of a charged residue in the hydrophobic face of TH1 abolishes cytotoxic activity, whereas replacement of a hydrophobic residue by a charged amino acid in the hydrophilic face of the helix has little, if any, effect on cytotoxic activity. In addition, we have identified Ser220 by site-directed mutagenesis as a residue that appears to be critical for correct folding of the fusion toxin. Mutations in this position result in fusion proteins that are extremely sensitive to proteolytic attack.
vanderSpek
J C
JC
Evans Department of Clinical Research, Boston University Medical Center, University Hospital, MA 02118.
Howland
K
K
Friedman
T
T
Murphy
J R
JR
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects; D004167:Diphtheria Toxin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity; D013329:Structure-Activity Relationship
D004167:Diphtheria Toxin; D000922:Immunotoxins; D007376:Interleukin-2; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C078456:denileukin diftitox