Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Peter, Marcus E.; Wittmann‐Liebold, Brigitte; Sprinzl, Mathias

doi:10.1021/bi00426a010

Cited by 39 publications

(22 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3a,b; retention time 28.21 min) was not radioactively labeled and could probably be generated by degradation or /3-elimination of the initial crosslinked reaction product [ 171. The same observation was made in affinity labeling experiments with T rhemophilus EF-Tu and oxidized GDP or GTP [7].…”

Section: R-s-y-g-i-p-y-i-e-t-s-a-kl47-t-r-q-g-v-el53supporting

confidence: 61%

“…This was, however, not attempted in the present investigation. In i 7 therinophilus EF-Tu the positions of covalent modification with oxidized GDP or GTP could be specified using solid-phase sequencing followed by measuring the I4C radioactivity in the soluble fractions after each sequencing step [7]. To analyze the amino acid positions of p21 cross-linked to [a-"P]GDP or [a-'*P]GTP, we tried to discover which lysine residues camed covalently bound "P-labeled guanosine nucleotides.…”

Section: N-terminal Edman Degradation Of Cross-linked Peptidesmentioning

confidence: 99%

See 1 more Smart Citation

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Löw

Sprinzl

Faulhammer

1993

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The amino acid sequence motifs of human c-H-rus p21 involved in the interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized [a-"P]GDP or [a-"P]GTP. Sitespecific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp-N digestion, HPLC separation of "P-labeled peptides and Nterminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of rus proteins, respectively, were identified. No significant divergences in the position and extent of covalent modification could be detected between p21 . GDP and p21 . GTP. This is in contrast to Thermus thermophilus EF-Tu . GDP and EF-Tu . GTP, which were investigated with the same technique [Peter, M. E., Wittmann-Liebold, M. (1988) Biochemistry 27,9132-91391 and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross-linking [a-3'P]GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.Among the superfamily of GTP-binding proteins [l], rus p21, the cellular homologue of the 21-kDa transforming proteins of Harvey and Kirsten murine sarcoma viruses (H-ms and K-rus), has become a prominent model for the study of the oncogenic potential of retroviruses, the role of transforming genes and the effects of somatic point mutations [2]. Considerable information on rus and rus-related proteins is now available with respect to amino acid sequence, threedimensional structure, post-translational modification, subcellular localization and the mechanism of guanosine nucleotide binding and hydrolase (GTPase) activity [3].Based on X-ray data, the architecture of the G-domain of c-H-rus p21 bound to various guanosine nucleotides and its interactions with these ligands have been defined in great detail and a mechanism of GTP hydrolysis has been proposed [4]. Comparison of the three-dimensional structures of p21 and the G-domains of bacterial elongation factor Tu (EFTu) [ 5 , 61 revealed a high degree of conserved topology and structural equivalence. Analysis of the nucleotide complexes of Escherichiu coli EF-Tu and human c-H-rus p21 further demonstrated that a conserved sequence motif, Asn-Lys-XaaAsp (N-K-X-D, where X stands for a variable amino acid) is involved in binding GDP or GTP.Recently, it could be shown that in Thermus rhermophilus EF-Tu or in a EF-Tu . EF-Ts complex part of this N-K-X-D sequence, the conserved K137, which is a component of the G-nucleotide binding site, reacts selectively with the aldehyde groups of periodate-oxidized GDP or GTP [7, 81. Later the same strategy was'applied to another GTPase, the human lymphocyte T-cell receptor [ chain [9]. Bovine trans...

show abstract

Section: R-s-y-g-i-p-y-i-e-t-s-a-kl47-t-r-q-g-v-el53supporting

confidence: 61%

Section: N-terminal Edman Degradation Of Cross-linked Peptidesmentioning

confidence: 99%

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Löw

Sprinzl

Faulhammer

1993

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Affinity labeling of EF-Tu with periodate-oxidized nucleotides was performed according to Peter et al [16]. For fast identification of the modified lysine residues after affinity labeling, the proteins were cleaved by trypsin (0.1 mg/ ml) for 30 min at 37"C, frozen, lyophilized and analyzed by 15% SDSiPAGE [23].…”

Section: Afinity Labeling Of Ef-tumentioning

confidence: 99%

“…GTPOxi, however, modifies both Lys52 and Lys137, with a preference for Lys137. Thus, the high reactivity of Lys137, compared to reactivity ofLysS2, is characteristic for the GTPinduced conformation of EF-Tu (Table 1) [16].…”

Section: Interaction Of Ef-tu With Ef-tsmentioning

confidence: 99%

“…Its amino acid sequence has been determined, revealing 70% sequence indentity to EF-Tu from E. coli [14, 151. Previously we were able to show by affinity labeling that Lys52 of T. thermophilus EF-Tu is in the vicinity of the bound nucleotide [16], although it is not part of one of the consensus elements typical for Gproteins [17].In this work we have used the affinity labeling of the GDP/ GTP binding site of EF-Tu from T. thermophilus in order to study the EF-Tu . EF-Ts interaction.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

Schirmer

Reiser

Sprinzl

1991

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDP,,,) or GTP (GTPoXi) in the absence and presence of elongation factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDP,,, and GTPnXi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTP,,,. GDP,,, reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., WittmanLiebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-91381. In the presence of EF-Ts, GDP,,, reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu . GDP,,, induces a conformation resembling that of the EF-Tu . GTP,,, complex in the absence of EF-Ts. Binding of EF-Ts to EFTu . GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu . GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA . EF -Tu . GTP coinplex formation. EF-Tu is a guanyl-nucleotide-binding protein (G-protein).This class of proteins includes the soluble proteins involved in polypeptide chain elongation (e. g. EF-Tu), the signal transducing G-proteins, proteins that control cell proliferation as well as those involved in polypeptide transport and secretion (for review see [I -41). The proteins of this superfamily resemble each other in their functional cycle; they exist in a GTP-bound active state (signal 'ON') or in a GDP-bound inactive state (signal 'OFF'). Oncogene product H-ras p21 is the only G-protein for which the three-dimensional structure in both conformations has been solved at atomic resolution by X-ray analysis [5, 61. Procaryotic EF-Tu is a well-characterized G-protein and thus a good candidate for structure/function studies of the interaction with other macromolecules; the effector [ribosome and aminoacyl tRNA (aatRNA) as coeffector] and the receptor (nucleotide-exchange factor EF-Ts). In its GTP form EFTu binds aatRNA and transports it to the bacterial ribosome. After GTP hydrolysis the binary EF-Tu . GDP complex is released from the ribosome. The release of GDP fromCorrespondence to M. Sprinzl, Laboratorium fur Biochemie der Universitat Bayreuth, Postfach 10 12 51, W-8580 Bayreuth, Federal Republic of Germany Ahhreviutions. EF-Tu, elongation h c t o r Tu; EF-TuRs9, elongation factor Tu cleaved at the Arg.59-Gly60 peptide bond; EF-Ts, elongation factor Ts; G-protein, guanyl-nucleotide-binding protein; aatRNA, aminoacyl transfer RNA; [AEDANS-s'C]Tyr-tRNATyr, Tyr-tRNA'IY' with penultimate 2-thiocytidine residue to which an N-(acetylaminoethyl)-5-naphthylamine-l -sulphonic acid is attached; GDP,,,, periodate-oxidized GDP; GTP,,,, pcriodate-oxidized GTP.Ensynzes. Pyruvatc kinase (EC 2.7.1.40); trypsin (EC 3.4...

show abstract

A Cell‐Physiological Description of G _E , A GTP‐Binding Protein that Mediates Exocytosis

Lillie

Gomperts

2007

Ciba Foundation Symposium 176 ‐ the GTPase Superfamily

View full text Add to dashboard Cite

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Cited by 39 publications

References 43 publications

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

A Cell‐Physiological Description of G _E , A GTP‐Binding Protein that Mediates Exocytosis

Contact Info

Product

Resources

About

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Cited by 39 publications

References 43 publications

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Affinity labeling of c‐H‐ras p21 consensus elements with periodate‐oxidized GDP and GTP

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

A Cell‐Physiological Description of G E , A GTP‐Binding Protein that Mediates Exocytosis

Contact Info

Product

Resources

About

A Cell‐Physiological Description of G _E , A GTP‐Binding Protein that Mediates Exocytosis