Asparagine‐135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

Weijland, Albert; Sarfati, Robert S.; Bârzu, Octavian; Parmeggiani, Andrea

doi:10.1016/0014-5793(93)80899-6

Cited by 5 publications

(3 citation statements)

References 28 publications

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“…2 Thus, this motif is important in interacting with G-protein-associated factors. Double mutants altering both Asn 135 and Asp 138 can completely inactivate nucleotide binding by EF1A, consistent with effects on affinity (11). Thus, the importance of these residues of the motif element on nucleotide binding is clear.…”

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confidence: 53%

Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange

Carr-Schmid¹,

Durko²,

Cavallius³

et al. 1999

Journal of Biological Chemistry

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A series of mutations in the highly conserved N 153 KMD 156 GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro. All three mutant proteins also show an increase in the K m for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1B␣ (eEF1B␣) and supported for growth by excess eEF1A was used to show the two mutations with the highest K m for GTP restore most but not all growth defects found in these eEF1B␣ deficient-strains to near wild type. An increase in K m alone, however, is not sufficient for suppression and may indicate eEF1B␣ performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo.

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confidence: 53%

Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange

Carr-Schmid¹,

Durko²,

Cavallius³

et al. 1999

Journal of Biological Chemistry

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“…The focus was residue D‐68, the final residue in the TVRD motif, because examples have been described where substitutions at this position have altered the substrate specificity of canonical GTPases [34,37,39–42]. In classical GTPases, the aspartate is considered to form a hydrogen bond with the amino group at position 2 of the guanine base, which is absent from the xanthine base [39,40]. Thus, D‐68 was substituted by asparagine and the hydrolytic activity of the TorD D68N variant towards GTP and XTP was measured using the malachite green assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

Intrinsic GTPase activity of a bacterial twin‐arginine translocation proofreading chaperone induced by domain swapping

et al. 2010

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The bacterial twin‐arginine translocation (Tat) system is a protein targeting pathway dedicated to the transport of folded proteins across the cytoplasmic membrane. Proteins transported on the Tat pathway are synthesised as precursors with N‐terminal signal peptides containing a conserved amino acid motif. In Escherichia coli, many Tat substrates contain prosthetic groups and undergo cytoplasmic assembly processes prior to the translocation event. A pre‐export ‘Tat proofreading’ process, mediated by signal peptide‐binding chaperones, is considered to prevent premature export of some Tat‐targeted proteins until all other assembly processes are complete. TorD is a paradigm Tat proofreading chaperone and co‐ordinates the maturation and export of the periplasmic respiratory enzyme trimethylamine N‐oxide reductase (TorA). Although it is well established that TorD binds directly to the TorA signal peptide, the mechanism of regulation or control of binding is not understood. Previous structural analyses of TorD homologues showed that these proteins can exist as monomeric and domain‐swapped dimeric forms. In the present study, we demonstrate that isolated recombinant TorD exhibits a magnesium‐dependent GTP hydrolytic activity, despite the absence of classical nucleotide‐binding motifs in the protein. TorD GTPase activity is shown to be present only in the domain‐swapped homodimeric form of the protein, thus defining a biochemical role for the oligomerisation. Site‐directed mutagenesis identified one TorD side‐chain (D68) that was important in substrate selectivity. A D68W variant TorD protein was found to exhibit an ATPase activity not observed for native TorD, and an in vivo assay established that this variant was defective in the Tat proofreading process. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7302371, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7302377: TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) and TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7302402: TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) and TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by comigration in non denaturing gel electrophoresis (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0404) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7302387: TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) and TorD (uniprotkb:http://www.uniprot.org/uniprot/P36662?format=text&ascii) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by cosedimentation in solution (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0028)

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“…For several classes of GTP-binding proteins, a strategy was devised where an aspartate to asparagine mutation shifted nucleotide specificity from guanine to xanthine ( Figure 1B). Examples include H-Ras (Zhong et al, 1995), EF-Tu (Hwang and Miller, 1987;Weijland et al, 1993), Ypt1 (Jones et al, 1995), Rab-5 (Hoffenberg et al, 1995;Rybin et al, 1996), FtsY (Powers and Walter, 1995), adenylosuccinate synthetase (Kang et al, 1994) and Goa (although here a second mutation was essential for shifting nucleotide specificity) (Yu et al, 1997). Xanthine nucleotides are only transiently produced during purine metabolism and are not populated as triphosphates in vivo, allowing for separation of the signaling effect of a specific GTPase in the background of other GTPases.…”

Section: Introductionmentioning

confidence: 99%

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

Strohmeier¹,

Hertel²,

Diederichsen³

et al. 2011

Biological Chemistry

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DEAD-box proteins disrupt or remodel RNA and protein/ RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo-and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in noncanonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the Ploop and other helicase signature motifs.

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Asparagine‐135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket

Cited by 5 publications

References 28 publications

Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange

Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange

Intrinsic GTPase activity of a bacterial twin‐arginine translocation proofreading chaperone induced by domain swapping

Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop

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