Localization of the sites of ADP‐ribosylation and GTP binding in the eukaryotic elongation factor EF‐2

Nilsson, Lars; Nygård, Odd

doi:10.1111/j.1432-1033.1985.tb08839.x

Cited by 39 publications

(25 citation statements)

References 38 publications

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“…Therefore, the toxin is able to bind eEF-2-GTP as it approaches the ribosome before forming the pre-translocation complex, as well as after it has departed the ribosome once GTP has been hydrolyzed. Earlier work using autoradiography had suggested that when NAD ϩ and diphtheria toxin (a catalytically similar toxin to ETA) is added to GTP-bound eEF-2, the factor is not ribosylated (32). However, the present work showed that the presence of bound guanyl nucleotides to eEF-2 did not alter the enzyme reaction rate compared with native eEF-2 (Table III).…”

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confidence: 53%

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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confidence: 53%

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Armstrong

Yates

Merrill

2002

Journal of Biological Chemistry

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“…The generated fragments were separated on SDS/polyacrylamide gradient gels containing 10-20% (w/v) acrylamide [4] and transferred to glassfiber filters as above. The stained filters were exposed to autoradiography for 3 weeks at -8O"C, and the fragmentation products were sequenced as above.…”

Section: Chemicalsmentioning

confidence: 99%

“…Oxidized GTP (oGTP) has been used as a reactive GTP analogue for affinity labelling of the GTP-binding domain of translational factors [4,131. The reagent contains two reactive aldehyde groups in the ribose ring, capable of reacting with neighbouring amino groups [14].…”

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confidence: 99%

Structural and functional studies of the interaction of the eukaryotic elongation factor EF‐2 with GTP and ribosomes

Nilsson

Nygård

1988

European Journal of Biochemistry

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The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at L~s '~l / L y s~~~.oGTP was covalently bound to the factor between Arg66 and L Y S~~' .After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/ Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.The eukaryotic elongation factor EF-2 is a single polypeptide chain with a molcular mass of 95 kDa [l]. The factor has at least three distinguishable functional properties. Guanosine nucleotides, GTP and GDP, form binary complexes with EF-2 [2], resulting in a conformational change in the factor, thereby adapting EF-2 for interaction with the ribosome [3, 41. The EF-2 . GTP complex associates with high affinity to ribosomes, having peptidyl-tRNA in the A-site, thereby promoting the translocation of the growing peptide chain from the A-site to the P-site [5 -71. The translocation is coupled to a hydrolysis of GTP to GDP and inorganic phosphate [8, 91. The catalytic domain, presumably located on the factor [lo], is activated by the interaction of the binary EF-2 . GTP complex with the ribosome [l 11. The post-translocation ribosomes formed have a reduced affinity for the factor [7], resulting in a facilitated release of the EF-2 . GDP complex during the ribosomal interaction with the ternary EF-1 . GTP

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“…It appears to be more likely that eubacterial EF-G is not modified by the action of diphtheria toxin, because the histidine residue at position 735 and its surrounding sequence is not present in the sequence of EF-G and shows no significant similarity to EF-2. ADP ribosylation of EF-2 reduces its affinity for the ribosome, leading to inhibition of protein synthesis [19]. lherefore, the carboxyl-terminal half is considered to contain the domain involved in the interaction of EF-2 with the ribosome and toxin.…”

Section: Protein Sequence Comparisonmentioning

confidence: 99%

Sequence analysis of the peptide‐elongation factor EF‐2 gene, downstream from those of ribosomal proteins H‐S12 and H‐S7, from the archaebacterial extreme halophile, Halobacterium halobium

Itoh

1989

European Journal of Biochemistry

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The gene for the peptide-elongation factor 2 (EF-2) was cloned from the archaebacterial extremc halophile Halobacterium halobium and sequenced. The 101 3 nucleotides upstream from this gene was two open rcading frames similar to ribosomal proteins S12 and S7 from Escherichiu coli. Sequence alignment studies showed the halobacterial elongation factor 2 to be equivalent to eukaryotic EF-2 and eubacterial EF-G. Sequcnce similarity to the eukaryotic elongation factor was much higher than to the eubacterial factor. Conserved sequence regions were present within the factor and are likely to constitute functionally important domains. These include the sites of GTP binding and ADP ribosylation by diphtheria toxin.The concept of archaebacteria as a third kingdom of organisms is based on comparative analyses of 16s rRNA [l].Protein synthesis machinery appears to be a promising subject for phylogenetic comparisons among the three kingdoms (eubacteria, archaebacteria and eukaryotes). Archaebacteria is a newly defined phylogenetic kingdom, characterized by a mixture of eubacterial, eukaryotic and unique archaebactcrial features [2-41. The peptide-elongation factor (EF-2 in eukaryote, EF-G in eubacteria), which catalyzes the translocation of-peptidyl-tRNA and mRNA on ribosomes, is essential for protein synthesis in all organisms. The archaebacterial elongation factor has the eukaryotic feature of sensitivity against ADP ribosylation by diphtheria toxin and a high degree of structrual similarity around the ADP -riboseaccepting site [5]. To determine how archaebacterial genes are transcribed and translated, and which phylogenetic relationships are present among the three kingdoms, the gene for the elongation factor from archaebacterial Halobacterium halobium was cloned and sequenced.The results indicated EF-2 from archaebacterial Halobacterium to be more similar to the eukaryotic EF-2 than to the eubacterial EF-G and the gene for the protein to lie downstream from those of ribosomal H-S12 and H-S7. This is analogous to the ribosomal protein S12 operon in E. coli.

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Localization of the sites of ADP‐ribosylation and GTP binding in the eukaryotic elongation factor EF‐2

Cited by 39 publications

References 38 publications

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A

Structural and functional studies of the interaction of the eukaryotic elongation factor EF‐2 with GTP and ribosomes

Sequence analysis of the peptide‐elongation factor EF‐2 gene, downstream from those of ribosomal proteins H‐S12 and H‐S7, from the archaebacterial extreme halophile, Halobacterium halobium

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