Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes

Sontag, B.; Reboud, Anne-Marie; Divita, Gilles; Pietro, Attilio Di; Guillot, Dominique; Reboud, Jean‐Paul

doi:10.1021/bi00059a014

Cited by 18 publications

(16 citation statements)

References 29 publications

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“…Initially, the binding of the nonhydrolyzable analogues of GDP/GTP to eEF-2 was evaluated by monitoring the quenching of the intrinsic fluorescence of eEF-2 upon titration of these nucleotides (data not shown). The decrease in fluorescence intensity of eEF-2 indicated an association of these nucleotides with eEF-2 at the saturating concentration of 50 M, which is in agreement with the earlier findings of Sontag et al (23). As Fig.…”

Section: Characterization Of the Single Cysteine Mutant Proteins Andsupporting

confidence: 93%

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Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Armstrong

Yates

Merrill

2002

Journal of Biological Chemistry

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The molecular nature of the protein-protein interactions between the catalytic domain from Pseudomonas aeruginosa exotoxin A (PE24H) and its protein substrate, eukaryotic elongation factor-2 (eEF-2) were probed using a fluorescence resonance energy transfer method. Single cysteine mutant proteins of PE24H were prepared and site-specifically labeled with the donor fluorophore IAEDANS (5-(2-iodoacetylaminoethylamino)-1-napthalenesulfonic acid), whereas eEF-2 was labeled with the acceptor fluorophore fluorescein. The association was found to be independent of ionic strength and of the co-substrate, NAD ؉ but dependent upon pH. The lack of requirement for NAD ؉ to produce the toxin-eEF-2 complex demonstrates that the catalytic process is a random order mechanism, thereby disputing the current model. The previously observed pH dependence for catalytic function can be assigned to the toxin-eEF-2 binding event, as the pH dependence of binding observed in this study showed a strong correlation with enzymatic activity. The ability of the toxin to bind eEF-2 with bound GTP/GDP was assessed using nonhydrolyzable analogues. The results from the substrate binding and catalytic activity experiments indicate that PE24H is able to interact and bind with eEF-2 in all of its guanyl nucleotide-induced conformational states. Thus, the toxin ribosylates eEF-2 regardless of the nucleotide-charged state of eEF-2. These results represent the first detailed characterization of the molecular details and physiological conditions governing this protein-protein interaction.

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Section: Characterization Of the Single Cysteine Mutant Proteins Andsupporting

confidence: 93%

“…nonhydrolyzable analogues in the absence of ribosomes (23). It was investigated whether the catalytic domain of ETA required a specific conformation of eEF-2 for protein-protein binding to occur.…”

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confidence: 99%

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Armstrong

Yates

Merrill

2002

Journal of Biological Chemistry

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“…The residues interacting with oxidized GTP were not identified. In our study of the quenching of eEF-2 intrinsic fluorescence by nucleotides, we could demonstrate an interaction of the factor not only with guanylic nucleotides, but also with adenylic ones (Sontag et al, 1993). The localization and significance of this interaction with adenylic nucleotides were not clarified.…”

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confidence: 69%

Photoaffinity Labeling of Elongation Factor‐2 with 8‐Azido Derivatives of GTP and ATP

Guillot

Yard

Reboud

1996

European Journal of Biochemistry

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Elongation factor 2 (eEF‐2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J. P. (1993) Biochemistry 32, 1976–1980]. Here we studied sites of these interactions by using photoactivable 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF‐2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH, poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non‐competitive inhibitors of the photoincorporation of 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP respectively. eEF‐2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N‐terminal sequencing. An octa‐peptide, Y264FDPANGK271, was the only peptide photolabeled with 8‐azido‐[γ‐32P]GTP whereas a N‐terminal fragment of about 7 kDa was the only one photolabeled with 8‐azido‐(γ‐32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF‐2.

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“…Fluorescence Measurements-These were performed using a SLM Aminco 8000C spectrofluorometer as described previously (17). The eEF-2 concentration used was 0.1 M, and the measurements were performed in a cuvette of 1 ml in 50 mM Tris-HCl, pH 7.4, 20 mM KCl, 10% (w/v) glycerol, 4 mM 2-mercaptoethanol.…”

Section: Methodsmentioning

confidence: 99%

A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

Vard

Guillot

Bargis

et al. 1997

Journal of Biological Chemistry

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The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli. They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis. The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them. When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits. Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not. Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form. These experiments reveal a distinct role for P1 and P2 in protein synthesis. Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein. This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2. P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.The large subunit of eukaryotic ribosomes contains 12-kDa acidic P proteins, which seem more numerous in lower eukaryotes than in higher organisms (1). In these, two types of P proteins are found, designated as P1 and P2 (2, 3). They share similar properties with prokaryotic proteins L7-L12, which form a pentamer with protein L10, (L7/L12) 4 -L10, constituting the lateral stalk of the 50 S ribosomal subunits (4). The eukaryotic P proteins, as their prokaryotic counterparts, seem to play an essential role in the interaction with elongation factors and in factor-dependent GTPase activity (5). However, some specific properties are observed with eukaryotic P proteins. First, these proteins exist on the ribosome as phosphorylated derivatives. They can be phosphorylated in vitro by either casein kinase II or by an endogenous ribosome-bound enzyme (6). Second, proteins P1-P2 present on the ribosome can exchange with a cytoplasmic pool of these unphosphorylated proteins (7). Phosphorylation of P1-P2, which appears to be necessary for ribosome activity, was originally suggested to be a requirement for the binding of these proteins to ribosomes (5), but recently this hypothesis has been challenged by new results obtained in yeast (8).We have shown previously that active rat liver 60 S ribosomal subunits could be reconstituted from inactive core particles prepared with 2,3-dimethylmaleic anhydride (DMMA) 1 (9), by adapting a method previously used with yeast ribosomes (10). Reactivation of the rat liver core particles was obtained not only with DMMA-split proteins containing several proteins including P1-P2 but also with a 50% ethanol, 0.08 M KCl extract containing P1-P2 exclusively. Dephospho...

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Intrinsic tryptophan fluorescence of rat liver elongation factor eEF-2 to monitor the interaction with guanylic and adenylic nucleotides and related conformational changes

Cited by 18 publications

References 29 publications

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Photoaffinity Labeling of Elongation Factor‐2 with 8‐Azido Derivatives of GTP and ATP

A Specific Role for the Phosphorylation of Mammalian Acidic Ribosomal Protein P2

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