Lipid interaction of diphtheria toxin and mutants

Abstract

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm).DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm).The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.Diphtheria toxin (DT) is responsible of most clinical symptoms of diphtheria [l]. It is a protein, produced by toxigenic strains of Corynebacterium diphtheriae as a single inactive polypeptide chain [2, 31. The toxin is activated by a specific proteolytic cleavage at an exposed loop that generates two chains held together by a single interchain disulfide bridge and by non-covalent forces [4]. Chain A (21 164 Da) ADPribosylates specifically elongation factor 2 of eukaryotes and archaebacteria, thus blocking protein synthesis [5]. Chain B (37 194 Da) binds via its carboxy-terminal region to a protein receptor [6, 71, but there is evidence that also acidic phospholipids may be involved in DT binding via headgroup interactions with both DT protomers [S, 91. After internalization, DT is included into intracellular acidic compartments, where by a mechanism yet unknown, chain A translocates across the membrane to reach the cytoplasm where it exerts its action [lo, 111. A key role in the intoxication process is played by toxin-lipid interactions because the toxin has to cross the lipid bilayer and also because of the suggested lipid involvement in DT cell binding.The interaction of DT with lipids has been studied with a variety of techniques (reviewed in [lo]). It is now clear that low pH induces a structural transition of DT, characterized by the exposure of hydrophobic surfaces and by an interaction with the fatty acid chains of phospholipids with a preference for acidic phospholipids. Both DT protomers are involved in this structural change, but at the present time the structural features of DT at acidic and neutral pH are poorly defined and data are lacking on the size of the part of DT that interacts with lipids and mediates the translocation of chain A in the In order to gain further insights on this poorly understood aspect of DT action we have used monolayers of both diphtheria toxin and lipids spread at the water-air interface and we have monitored the lipid interaction of DT by surfa...

The adsorption of Pseudomonas aeruginosa exotoxin A to phospholipid monolayers is controlled by pH and surface potential

“…This value is -50% larger than that found for cardiotoxin, a molecule of m.w. 7000 (Bougis et al, 1981), but only one-fourth that of colicin A (Pattus et al, 1983), and much smaller than that of DT (-30 nm2; Demel et al, 1991). This suggests that only a small portion of the ETA molecule is actually inserted into the lipid monolayer.…”

Ion channel and membrane translocation of diphtheria toxin

“…The structural changes of DT-induced by pH in relation to model membranes have been studied with a variety of techniques including liposome binding [34], membrane photolabelling with photoreactive lipids [35][36][37] and phospholipids [38,39], circular dichroism [36,40], tryptophan fluorescence [41,42], liposome fusion and leakage [42][43][44][45][46], scanning calorimetry [47], polarized infrared spectroscopy [48] and lipid monolayers [49]. Notwithstanding the differences among the experimental approaches, these studies show a substantial agreement on the main findings with a good correlation with results obtained with cellular systems.…”

Computer modelling of the transmembrane channel formed by a CNBr peptide of diphtheria toxin B fragment

“…After endocytosis, these toxins are exposed to acidic pH within the endosomal lumen (pH 5-6), which triggers translocation. For DT, acidic pH causes the toxin to undergo a conformational change that exposes cryptic hydrophobic sequences in its B fragment (DTB 342 residues) and promotes insertion of the toxin into membranes [4][5][6][7][8][9][10][11]. After membrane insertion, DTA is released into the cytosol [12].…”