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Query: UNIPROT:P06889 (Mol)
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Crystals of the dimeric aspartyl-tRNA synthetase from Escherichia coli (molecular mass 132,000 Da) complexed with its cognate tRNA (molecular mass 25,000 Da) have been grown using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group C222(1) with unit cell parameters a = 102.75 A, b = 128.11 A, c = 231.70 A and diffract to 3 A. The asymmetric unit contains one monomer of the aspartyl-tRNA synthetase and one tRNA molecule.
J Mol Biol 1992 Apr 20
PMID:Crystallization of aspartyl-tRNA synthetase-tRNA(Asp) complex from Escherichia coli and first crystallographic results. 156 73

The structure and function of in vitro transcribed tRNA(Asp) variants with inserted conformational features characteristic of yeast tRNA(Phe), such as the length of the variable region or the arrangement of the conserved residues in the D-loop, have been investigated. Although they exhibit significant conformational alterations as revealed by Pb2+ treatment, these variants are still efficiently aspartylated by yeast aspartyl-tRNA synthetase. Thus, this synthetase can accommodate a variety of tRNA conformers. In a second series of variants, the identity determinants of yeast tRNA(Phe) were transplanted into the previous structural variants of tRNA(Asp). The phenylalanine acceptance of these variants improves with increasing the number of structural characteristics of tRNA(Phe), suggesting that phenylalanyl-tRNA synthetase is sensitive to the conformational frame embedding the cognate identity nucleotides. These results contrast with the efficient transplantation of tRNA(Asp) identity elements into yeast tRNA(Phe). This indicates that synthetases respond differently to the detailed conformation of their tRNA substrates. Efficient aminoacylation is not only dependent on the presence of the set of identity nucleotides, but also on a precise conformation of the tRNA.
J Mol Biol 1992 Jul 20
PMID:Effect of conformational features on the aminoacylation of tRNAs and consequences on the permutation of tRNA specificities. 164 Apr 53

Three new crystal forms of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase have been produced. The best crystals, obtained after modifying both purification and crystallization conditions, belong to space group P2(1)2(1)2(1) and diffract to 2.7 A. Unit cell parameters are a = 210.4 A, b = 145.3 A and c = 86.0 A (1 A = 0.1 nm), with one dimeric enzyme and two tRNA molecules in the asymmetric unit.
J Mol Biol 1988 May 05
PMID:A high resolution diffracting crystal form of the complex between yeast tRNAAsp and aspartyl-tRNA synthetase. 304 97

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.
J Mol Biol 1985 Aug 05
PMID:Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea. 390 Apr 15

A large scale purification procedure of baker's yeast aspartyl-tRNA synthetase is described which yields more than 200 mg pure protein starting from 30 Kg of wet commercial cells. The synthetase is an alpha 2 dimer of Mr = 125,000 +/- 5,000 which can be crystallized (J. Mol. Biol. 138, 1980, 129-135). The enzyme has an elongated shape with a Stokes radius of 50 A and a frictional ratio of 1.5. The synthetase has a tendency to aggregate but methods are described where this effect is overcome.
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PMID:Large scale purification and structural properties of yeast aspartyl-tRNA synthetase. 636 67

A protein domain corresponding to residues 31 to 149 of the E. coli Lysyl-tRNA synthetase species corresponding to the lysS gene was expressed and 15N-labelled. 1H and 15N NMR resonance assignments for this domain were obtained by two-dimensional and three-dimensional homonuclear and heteronuclear spectroscopy. Using distance geometry and simulated annealing, a three-dimensional structure could be calculated using 701 NOE and 86 dihedral angle restraints. It is composed of a five-stranded antiparallel beta-barrel capped by three alpha-helices at its ends. This structure closely resembles that of the N-terminal domain of the other E. coli lysyl-tRNA synthetase species expressed from the lysU gene and is highly homologous to the fold observed for the corresponding region of aspartyl-tRNA synthetase. It is shown that the isolated N-terminal fragment of lysyl-tRNA synthetase can interact with tRNA(Lys) as well as with poly (U), which mimics the anticodon sequence. Amino acid residues involved in these interactions were identified and, in the case of poly-U, a number of specific protein-RNA contacts were characterized. Specific recognition of tRNA(Lys) involves a cluster of four structurally well-defined aromatic residues, anchored on the beta-strands, and basic residues located on the surrounding loops. This organization is reminiscent of other RNA binding proteins, such as the U1A small nuclear ribonucleoprotein.
J Mol Biol 1995 Oct 13
PMID:Solution structure of the anticodon-binding domain of Escherichia coli lysyl-tRNA synthetase and studies of its interaction with tRNA(Lys). 747 6

Previous sequence analyses have suggested the existence of two distinct classes of aminoacyl-tRNA synthetase. The partition was established on the basis of exclusive sets of sequence motifs (Eriani et al. [1990] Nature 347:203-306). X-ray studies have now well defined the structural basis of the two classes: the class I enzymes share with dehydrogenases and kinases the classic nucleotide binding fold called the Rossmann fold, whereas the class II enzymes possess a different fold, not found elsewhere, built around a six-stranded antiparallel beta-sheet. The two classes of synthetases catalyze the same global reaction that is the attachment of an amino acid to the tRNA, but differ as to where on the terminal adenosine of the tRNA the amino acid is placed: class I enzymes act on the 2' hydroxyl whereas the class II enzymes prefer the 3' hydroxyl group. The three-dimensional structure of aspartyl-tRNA synthetase from yeast, a typical class II enzyme, is described here, in relation to its function. The crucial role of the sequence motifs in substrate binding and enzyme structure is high-lighted. Overall these results underline the existence of an intimate evolutionary link between the aminoacyl-tRNA synthetases, despite their actual structural diversity.
J Mol Evol 1995 May
PMID:The class II aminoacyl-tRNA synthetases and their active site: evolutionary conservation of an ATP binding site. 778 25

Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.
Mol Cell Biochem 1994 Nov 09
PMID:Expression of human aspartyl-tRNA synthetase in COS cells. 787 98

The crystal structures of Thermus thermophilus aspartyl-tRNA synthetase and of its complex with ATP, Mg2+ and aspartic acid, show in situ formation of the amino acid adenylate and furnish experimental evidence for the modes of recognition of aspartic acid and ATP. The amino acid fits in a predefined specific site in which it replaces water molecules without significant conformational changes of the binding residues. This mode of selection is reminiscent of the lock and key concept. The pocket is closed by the movement of a histidine side chain from a neighbouring loop acting as a valve. ATP binding is driven by the stacking of the adenine upon the otherwise fixed aromatic ring of the class-II-invariant phenylalanine Phe235. Specific recognition is achieved by interactions with the flexible side chains of other class-II-conserved residues. Conformational changes have been identified which allow the description of a reaction pathway including both lock-and-key and induced-fit interactions. This pathway can presumably be extended to all class II aaRS.
J Mol Biol 1994 Nov 25
PMID:Synthesis and recognition of aspartyl-adenylate by Thermus thermophilus aspartyl-tRNA synthetase. 796 28

Specific amino acid binding by aminoacyl-tRNA synthetases is necessary for correct translation of the genetic code. To obtain insight into the origin of the specificity, the binding to aspartyl-tRNA synthetase (AspRS) of the negatively charged substrate aspartic acid and the neutral analogue asparagine are compared by use of molecular dynamics and free energy simulations. Simulations of the Asn-AspRS complex show that although Asn cannot bind in the same position as Asp, several possible positions exist 1.5 to 2 A away from the Asp site. The binding free energy of Asn in three of these positions was compared to that of Asp through alchemical free energy simulations, in which Asp is gradually mutated ito Asn in the complex with the enzyme. To correctly account for the electrostatic interactions in the system (including bulk solvent), a recently developed hybrid approach was used, in which the region of the mutation site is treated microscopically, whereas distant protein and solvent are treated by continuum electrostatics. Seven free energy simulations were performed in the protein and two in solution. The various Asn positions and orientations sampled at the Asn endpoints of the protein simulations yielded very similar free energy differences. The calculated Asp-->Asn free energy change is 79.8(+/-1.5) kcal/mol in solution and 95.1(+/-2.8) kcal/mol in the complex with the protein. Thus, the substrate Asp is predicted to bind much more strongly than Asn, with a binding free energy difference of 15.3 kcal/mol. This implies that erroneous binding of Asn by AspRS is highly improbable, and cannot account for any errors in the translation of the genetic code. Almost all of the protein contributions to the Asp versus Asn binding free energy difference arise from an arginine and a lysine residue that hydrogen bond to the substrate carboxylate group and an Asp and a Glu that hydrogen bond to these; all four amino acid residues are completely conserved in AspRSs. The protein effectively "solvates" the Asp side-chain more strongly than water does. The simulations are analyzed to determine the interactions that Asn is able to make in the binding pocket, and which sequence differences between AspRS and the highly homologous AsnRS are important for modifying the amino acid specificity. A double or triple mutation of AspRS that could make it specific for Asn is proposed, and supported by preliminary simulations of a mutant complex.
J Mol Biol 1998 Feb 06
PMID:Specific amino acid recognition by aspartyl-tRNA synthetase studied by free energy simulations. 948 Jul 72


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