The N-terminal alpha-helix of fragment B of diphtheria toxin promotes translocation of fragment A into the cytoplasm of eukaryotic cells.
Diphtheria toxin consists of two parts, fragments A and B. Fragment A has enzymatic activity inhibiting protein synthesis. Fragment B binds to cellular receptors and, upon exposure to low pH, inserts into the membrane, forms cation-selective channels, and facilitates translocation of fragment A. Previous data have suggested that the N-terminal part of fragment B, including the amphipathic alpha-helix TH1, plays an active role during translocation of fragment A (Madshus, I. H., Wiedlocha, A., and Sandvig, K. (1994) J. Biol. Chem. 269, 4648-4652). When replacing charged residues in TH1 with uncharged amino acids, translocation of fragment A was strongly inhibited, virtually without affecting binding of the toxin or channel activity. These data suggest that TH1 may act as a targeting/anchoring sequence. In a mutant with eight positive charges and one negative charge in TH1, increased specific binding was observed, even if TH1 was outside the toxin's binding domain. This suggests that TH1 could be important in binding to parts of the translocation machinery. Fragment A associated with this mutant fragment B was translocated 10-fold more efficiently than wild-type toxin. The fact that this mutant TH1 efficiently promoted translocation, while, a hydrophobic TH1 did not, suggests that TH1 does not interact with the hydrophobic part of the membrane phospholipids.
Madshus
I H
IH
Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003593:Cytoplasm / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D011487:Protein Conformation; D014709:Vero Cells
D004167:Diphtheria Toxin; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; C039171:diphtheria toxin fragment B