Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin

Burns, Gary R.; Abraham, Abraham K.; Vedeler, Anni

doi:10.1016/0014-5793(86)81021-3

Cited by 18 publications

(11 citation statements)

References 15 publications

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“…The similar K D values for GTP and GDP binding to yeast eEF2 are in contrast to previous results for both prokaryotic EF-G and eukaryotic eEF2 in the absence of ribosomes where the K D for GDP binding is significantly lower than for GTP (53,54). In addition, our observation that the binding affinity of eEF2 for GTP and GDP is unaffected by ADP-ribosylation disagrees with previous results showing the affinity for GTP to wheat germ ADPR-eEF2 was significantly reduced (32).…”

Section: Discussioncontrasting

confidence: 57%

“…This could allow initial binding and GTP hydrolysis, which has been shown for EF-G to be a very early event in translocation (5). Other reports have indicated a marked decrease in the ability of ADPR-eEF2 to associate with the 80 S ribosome (32)(33)(34)(35) as well as a significant reduction in binding and hydrolysis of GTP upon ADP-ribosylation (32,35). Furthermore, ADPR-eEF2 binds weaker than eEF2 to a synthetic oligonucleotide (SRD RNA) mimicking the sarcin/ricin loop of 28 S ribosomal RNA (51).…”

Section: Discussionmentioning

confidence: 99%

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Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae

Jørgensen

Yates

Teal

et al. 2004

Journal of Biological Chemistry

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The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 Å resolution. The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor. We have obtained an electron density map of ADP-ribosylated translation factor 2 revealing both the ADP-ribosylation and the diphthamide. This is the first structure showing the conformation of an ADP-ribosylated residue and confirms the inversion of configuration at the glycosidic linkage. Binding experiments show that the ADP-ribosylation has limited effect on nucleotide binding affinity, on ribosome binding, and on association with exotoxin A. These results provide insight to the inhibitory mechanism and suggest that inhibition may be caused by erroneous interaction of the translation factor with the codon-anticodon area in the P-site of the ribosome.

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Section: Discussioncontrasting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae

Jørgensen

Yates

Teal

et al. 2004

Journal of Biological Chemistry

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“…However, others have shown that ADP R -eEF2 displays a specific defect in binding to pretranslocation ribosomes (21). Furthermore, early studies reported that ADP R -eEF2 showed a decrease in GTP binding (22) whereas more recent fluorescent analyses showed no difference in GTP binding by ADP R -eEF2 (18). Taken together, these biochemical experiments suggest that ADP R -eEF2 is able to bind GTP and the ribosome but is unable either to efficiently or accurately promote translocation.…”

mentioning

confidence: 55%

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Mateyak

Kinzy

2013

Journal of Biological Chemistry

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Background: Diphtheria toxin inhibits translation by ADP-ribosylation of elongation factor 2. Results: Diphtheria toxin expression results in accumulation of cells that fail to separate following mitosis. Conclusion: Diphtheria toxin expression has unique effects on translocation and the production of specific proteins. Significance: Understanding how ADP-ribosylated elongation factor 2 affects translocation will increase knowledge of translation and potentially lead to new treatments for toxin exposure.

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“…The mutant toxins were also examined for changes in conformational state as a function of pH, using the fluorescent probe TNS, which exhibits a higher quantum yield in nonpolar than in polar media (Burns et al, 1986;Collins & Collier, 1987). Mutant or $wt toxin (100 nM) was incubated with TNS (150 pM) for 15 min at room temperature in buffers at varying pH before readings were taken.…”

Section: Bmentioning

confidence: 99%

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin

et al. 1996

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Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH 5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A+D352A and fourfold for E349Q+D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.Keywords: diphtheria toxin; membrane insertion; membrane translocation; pH dependence Many protein toxins undergo direct interactions with the plasma membrane or membranes of internal compartments of their target cells. Some form channels in the plasma membrane and ultimately cause colloid osmotic lysis, whereas others undergo

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Nucleotide binding to elongation factor 2 inactivated by diphtheria toxin

Cited by 18 publications

References 15 publications

Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae

Crystal Structure of ADP-ribosylated Ribosomal Translocase from Saccharomyces cerevisiae

ADP-ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin

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