title: Mechanism of insertion of diphtheria toxin: peptide entry and pore size determinations.
abstract: Diphtheria toxin ( DTx ) is an extremely potent inhibitor of protein synthesis. It is secreted as a linear polypeptide, which is cleaved to produce disulfide-linked A and B fragments. Fragment A, the inhibitor of protein synthesis, requires fragment B, the recognition subunit, for entry into intact cells. Fragment B has been proposed to form a transmembrane channel through which A gains access to the cytosol. If it were demonstrated that the B subunit had an exclusive association with membrane lipid acyl chains, this might indicate that A is secluded in a proteinaceous B channel. However, our results from intramembranous photolabeling studies show that both subunits of DTx enter the hydrocarbon domain of the bilayer. Toxin cleavage is not required for penetration. Decreasing pH leads to increased binding and hence indirectly to increased penetration. Parallel permeability studies indicate that cleaved DTx does indeed form pores (24 A in diameter) and they are larger than those previously reported (5 A) with native toxin. The data suggest that these are dimeric structures. Cleaved DTx is much more effective than intact DTx at pore formation. Thus, we conclude that, while pore formation is a feature of toxin-membrane interaction, the pore structure does not protect A from contact with lipid side chains and may in fact consist of both the A and B domains in a dimeric configuration, (AB)2.
mesh_terms: D001692:Biological Transport; D004167:Diphtheria Toxin / Q000378:metabolism*; D006017:Glycolipids; D066257:Heparin-binding EGF-like Growth Factor; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins / Q000378:metabolism; D010222:Parainfluenza Virus 1, Human; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004167:Diphtheria Toxin; D006017:Glycolipids; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011950:Receptors, Cholinergic