Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation

Jiang, Jean X.; Abrams, Franklin S.; London, Erwin

doi:10.1021/bi00230a008

Cited by 43 publications

(35 citation statements)

References 56 publications

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“…Quenching by Anti-BODIPY Antibodies-Anti-BODIPY experiments were done using sonicated small unilamellar vesicles prepared as de-scribed previously (17). Mixtures containing 1 mol of total lipid composed of 70% DMoPC and 30% DOPG or composed of 70% DOPC and 30% DOPG (mol/mol) were prepared.…”

Section: Effect Of Increasing T Domain Concentration On Bimane Fluorementioning

confidence: 99%

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Identification of Shallow and Deep Membrane-penetrating Forms of Diphtheria Toxin T Domain That Are Regulated by Protein Concentration and Bilayer Width

Wang¹,

Malenbaum²,

Kachel³

et al. 1997

Journal of Biological Chemistry

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190

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The ␣-helix-rich, hydrophobic transmembrane (T) domain of diphtheria toxin is believed to play a central role in membrane insertion by the toxin and in the translocation of its catalytic domain across membranes. In this report, T domain structure was studied using site-directed single-Cys mutants. The residues chosen, 322 (near the amino-terminal end of helix TH8), 333 (within helix TH8), and 356 (within helix TH9) were substituted with Cys and labeled with the fluorescent probe bimane. (Residues 333 and 356 should be located within the bilayer in the transmembrane state, and residue 322 should not penetrate the bilayer.) After insertion of T domain into model membrane vesicles, the location of bimane label relative to the lipid bilayer was characterized by its fluorescence emission and by its quenching with nitroxide-labeled phospholipids. It was found that when the T domain is added to dioleoylphosphatidylcholine-containing vesicles, all three residues reside close to the outer surface. However, at high T domain concentration or in thinner dimyristoleoylphosphatidylcholine-containing vesicles, a large fraction of residues 333 and 356 penetrate deeply into the membrane. In contrast, residue 322 remains exposed to aqueous solution under these conditions. These conclusions were confirmed by a novel antibody binding method. Antibodies that quench the fluorescence of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacene (BODIPY) groups were used to evaluate the exposure of BODIPYlabeled 322, 333, and 356. Maximum exposure of residues 333 and 356 to externally added antibody was only observed under conditions in which bimane fluorescence showed that these residues do not penetrate the bilayer. In contrast, residue 322 remained exposed under all conditions. We propose that the deeply penetrating T domain conformation represents a transmembrane or near-transmembrane state. The regulation of the transmembrane/nontransmembrane equilibrium should be a key to understanding diphtheria toxin membrane insertion and translocation. Our results suggest that toxintoxin interactions may play an important role in regulating this behavior.Diphtheria toxin is a protein secreted by Corynebacterium diphtheriae. It can be split into two chains, A (21 kDa) and B (37 kDa), joined by a disulfide bond (1). The crystal structure of the toxin shows that it consists of three domains (2-5). The A chain is the catalytic (C) domain. The B chain contains the transmembrane (T) and receptor binding (R) domains. Membrane penetration is believed to occur after the toxin reaches endosomes (6). The low pH within the endosomal lumen induces a partial unfolding of the toxin, resulting in exposure of the hydrophobic regions and translocation of the A chain of the toxin into the cytoplasm (6). Once in the cytoplasm, the A chain catalyzes the transfer of the ADP-ribosyl group of NAD ϩ to elongation factor 2, inactivating protein synthesis.The T domain is made up of nine ␣-helices, several of which contain hydrophobic sequences that play a critical role in...

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Section: Effect Of Increasing T Domain Concentration On Bimane Fluorementioning

confidence: 99%

“…The mixed lipids were dried under an N 2 stream and then redissolved with 7.5 l of ethanol. Vesicles were prepared by rapidly diluting this solution with 300 l of 500 mM sodium acetate, 150 mM NaCl, pH 4.4 (17). 2 Then 5 g of labeled T domain mutant in 300 l of Tris-Cl, pH 8, was added, yielding a final pH 4.5.…”

mentioning

confidence: 99%

Identification of Shallow and Deep Membrane-penetrating Forms of Diphtheria Toxin T Domain That Are Regulated by Protein Concentration and Bilayer Width

Wang¹,

Malenbaum²,

Kachel³

et al. 1997

Journal of Biological Chemistry

Self Cite

190

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“…Although the effects of pH on the conformation have been studied by various methods, there is little information available about the precise mechanism of triggering by acidic conditions (8,19). The current mutant search was undertaken in hopes of identifying one or more specific amino acid residues within DTB that are involved in triggering the acidic-pH-dependent conformational change and translocation.…”

Section: Introductionmentioning

confidence: 99%

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.

O'Keefe

Cabiaux

Choe

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm ofE. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. cofl were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349 --Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytoxicity and membrane translocation in mammalian cells and lethality for E. col but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic a-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.Bacterial proteinaceous toxins that insert into and traverse membranes offer interesting opportunities to study the interactions of proteins with lipid bilayers. Diphtheria toxin (DT, 535 residues) belongs to a class of toxins that enzymically modify substrates within mammalian cells (1). Many of these toxins are initially taken into cells by receptor-mediated endocytosis, and subsequently, an enzymically active polypeptide moiety of the toxin is released into the cytosol from endosomes or another membrane-bound compartment. For DT, a proteolytic fragment (fragment A, or DTA) is transferred to the cytosol and catalyzes the ADP-ribosylation of elongation factor 2 and thereby inhibits protein synthesis and causes death of the cell (2). How the enzymically active moiety crosses a membrane is not well-understood for any toxin but, for DT and certain other toxins (and for many animal viruses, as well), acidic intravesicular pH is known to trigger the process. A variety of evidence indicates that the membrane translocation event for DT occurs when the toxin is exposed to a pH near 5.0 in the endosomal compartment (3-6). In vitro, treatment of DT with buffer of pH near 5.0 induces a conformational change that causes the toxin to insert into artificial lipid bilayers (7-10), as manifested, for example, by the formation of ion-conductive m...

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“…These toxins first bind to the receptors on the cell membrane, which triggers the membrane-mediated endocytosis. The low pH condition of the acidic organelles in endocytic pathway, such as the endosome, can induce the membrane insertion of internalized toxins, which might facilitate the release of the catalytic A chain of the toxin into the cytoplasm (37). But the membrane translocation mechanism in detail remains unsolved.…”

Section: Discussionmentioning

confidence: 99%

Trichosanthin Induces Leakage and Membrane Fusion of Liposome

2003

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SummaryTrichosanthin (TCS) is a ribosome inactivating protein with multiple pharmacological properties. Here the interaction between TCS and a phospholipid bilayer is investigated to provide evidence for membrane translocation mechanism of TCS. The results show that TCS can destabilize liposomes made by phospholipids with negatively charged head group. The destabilization effect is pHdependent and happens only under acidic conditions. Membrane fusion is also seen to accompany the destabilizing process. The interaction between a phospholipid bilayer and C7, a mutant of TCS with 7 residues at its C-terminus deleted, has been investigated. Deleting the C-terminus almost completely abolishes the destabilizing effect of TCS on the phospholipid bilayer, which implicates the C-terminus in the interaction between trichosanthin and the membrane. IUBMB Life, 55: 681-687, 2003

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Folding changes in membrane-inserted diphtheria toxin that may play important roles in its translocation

Cited by 43 publications

References 56 publications

Identification of Shallow and Deep Membrane-penetrating Forms of Diphtheria Toxin T Domain That Are Regulated by Protein Concentration and Bilayer Width

Identification of Shallow and Deep Membrane-penetrating Forms of Diphtheria Toxin T Domain That Are Regulated by Protein Concentration and Bilayer Width

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.

Trichosanthin Induces Leakage and Membrane Fusion of Liposome

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