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D Moras

Publications and source records attributed to D Moras.

At least 145 records · Page 8Linked to original sources
PubMed1993

Yeast tRNA(Asp) recognition by its cognate class II aminoacyl-tRNA synthetase.

Aminoacyl-RNA synthetases can be divided into two classes according to structural features inferred from sequence alignments. This classification correlates almost perfectly with the attachment of the amino acid to the 2'-OH (class I) or 3'-OH (class II) group of the terminal adenosine. Six subgroups of higher homology can be inferred from sequence analysis. The five aminoacyl-tRNA synthetases whose crystal structures are known (MetRS, TyrRS and GlnRS in class I, SerRS and AspRS in class II) belong to different subgroups. Two of them, GlnRS and AspRS, have been cocrystallized with their cognate tRNA. AspRS, like six other members of class II, is an alpha 2 dimer. Yeast tRNA(Asp) exhibits five identity determinants: the three anticodon bases, the discriminator base G73 and the base pair G10-U25. We report here that the refined crystal structure of AspRS complexed with tRNA(Asp) at 2.9 A resolution reveals three regions of contact, each involving a domain of AspRS and at least one identity determinant of tRNA(Asp). The mode of binding of the acceptor stem of tRNA(Asp) by AspRS can be generalized to class II aminoacyl-tRNA synthetases, whereas the deciphering of the anticodon, which involves a large conformational change of the loop and the formation of a bulge, is more specific to the aspartic system.

Amino Acid Sequence↗
PubMed1993

Structure determination of aldose reductase: joys and traps of local symmetry averaging.

The structure of aldose reductase, a monomeric enzyme of 314 amino acids which crystallizes in space group P1 with four monomers per asymmetric unit, has been solved using a combination of single isomorphous replacement (SIR), solvent flattening and local symmetry averaging. The self rotation showed evidence of 222 local symmetry. The map calculated from the original single isomorphous replacement phases showed a clear solvent envelope but was uninterpretable. A first averaging attempt failed because the molecular envelope obtained from the SIR map weighted with monomer correlation was too small and the averaging was biased by low-resolution truncation. A second attempt with an enlarged envelope and including low-resolution reflections succeeded in refining phases at 3.5 A resolution but failed to extend them correctly. Rigid-body refinement of a partial model based on the 3.5 A map calculated from refined phases showed significant departures from the 222 symmetry. A third averaging attempt using the improved symmetry succeeded in producing a clear map with phases extended to 3.07 A resolution. This map revealed a (beta/alpha)(8) fold, not previously found in NADPH-dependent enzymes. This work shows the importance of mask definition and local symmetry elements accuracy for averaging, and describes a method for improving these parameters.

Journal Article↗
PubMed1993

The aminoacyl-tRNA synthetase family: modules at work.

The combined use of molecular and structural biology techniques has proved very efficient in elucidating structure-function relationships in aminoacyl-tRNA synthetases. Our present understanding of this family of enzymes is based on two main unifying principles: (i) division into two different classes, corresponding to two different modes of ATP binding and attachment of the activated amino acid to the last nucleotide of tRNA (either 2'OH or 3'OH of the ribose) by two different catalytic mechanisms and two structural domains with completely different folding, and (ii) the modular organization into separate and additional domains that we are just beginning to understand. Sequence analysis complements very nicely existing structural, biochemical and genetic results and makes them more general, leading to verifiable predictions.

Amino Acid Sequence↗
PubMed1993

Yeast aspartyl-tRNA synthetase: a structural view of the aminoacylation reaction.

The refinement of the crystal structure of a binary complex formed by yeast AspRS and tRNA(Asp) provided a detailed understanding of the recognition of tRNA by an aminoacyl-tRNA synthetase. The crystal structures of several complexes containing ATP, alone or with aspartic acid, were also determined and refined. These studies led to a complete description of the active site of the enzyme and to the elucidation of the location and interactions of the various substrates. Based on these structural results, a class II-specific pathway for the aminoacylation reaction can be proposed.

Acylation↗
PubMed1993

Structural aspects and evolutionary implications of the recognition between tRNAs and aminoacyl-tRNA synthetases.

Recent structural discoveries in the fields of tRNAs and aminoacyl-tRNA synthetases (aaRS), such as the existence of two classes of aminoacyl tRNA synthetases, the modular character of aaRS with domains homologous to other existing proteins, and the absence of direct interactions between the catalytic site containing the aminoacid binding pocket and the anticodon point the way to an understanding of the origin of the genetic code.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Recognition of tRNAs by aminoacyl-tRNA synthetases.

Our present understanding of the molecular mechanisms responsible for the recognition of tRNAs by their cognate aminoacyl-tRNA synthetases (aaRS) is essentially based on three sources of information: 1) the characterization of tRNA identity determinants using in vivo and in vitro approaches, 2) the classification of synthetases from primary sequence analysis: aaRS can be partitioned into two classes according to the spatial structure of their ATP binding domain, and 3) the structural results of crystallographic investigations and solution studies. The crystal structures of three aaRS and two complexes, one of each class, are known to atomic resolution. tRNA recognition has two structural components. The interaction between the acceptor end and the active site domain is class-specific and the binding mode of the stem observed in the crystal structures of GlnRS-tRNA(Gln) and AspRS-tRNA(Asp) complexes can be generalized to their respective classes. Identity determinants located in other parts of the tRNA molecule are decoded by different domains of the enzyme. These protein modules exhibit a large structural diversity. The recognition process is then system or subgroup specific.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Three-dimensional structure of phenylalanyl-transfer RNA synthetase from Thermus thermophilus HB8 at 0.6-nm resolution.

The three-dimensional structure of the heterodimeric alpha 2 beta 2 enzyme phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 has been determined by X-ray crystallography, using the multiple-isomorphous-replacement method at 0.6 nm resolution. Trigonal crystals of space group P3(2)21 have cell dimensions a = b = 17.6 nm and c = 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V = 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation-function calculation and measurement of the density of the native crystals, we postulate the existence of only the alpha beta dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy-atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent-flattening procedure were used for electron-density-map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with alpha beta dimer only in the asymmetric unit. The phenylalanyl-tRNA synthetase from T. thermophilus molecule has a 'quasi-linear' subunit organization. As can be concluded at this level of resolution, there is no contact between small alpha subunits in the functional heterodimer.

Chemical Phenomena↗
PubMed1992

Crystallization of aspartyl-tRNA synthetase-tRNA(Asp) complex from Escherichia coli and first crystallographic results.

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.

Aspartate-tRNA Ligase↗
PubMed1992

Novel NADPH-binding domain revealed by the crystal structure of aldose reductase.

Aldose reductase is the first enzyme in the polyol pathway and catalyses the NADPH-dependent reduction of D-glucose to D-sorbitol. Under normal physiological conditions aldose reductase participates in osmoregulation, but under hyperglycaemic conditions it contributes to the onset and development of severe complications in diabetes. Here we present the crystal structure of pig lens aldose reductase refined to an R-factor of 0.232 at 2.5-A resolution. It exhibits a single domain folded in an eight-stranded parallel alpha/beta barrel, similar to that in triose phosphate isomerase and a score of other enzymes. Hence, aldose reductase does not possess the expected canonical dinucleotide-binding domain. Crystallographic analysis of the binding of 2'-monophospho-adenosine-5'-diphosphoribose, which competitively inhibits NADPH binding reveals that it binds into a cleft located at the C-terminal end of the strands of the alpha/beta barrel. This represents a new type of binding for nicotinamide adenine dinucleotide coenzymes.

Aldehyde Reductase↗
PubMed1992

Rapid purification of the Aeromonas proteolytica aminopeptidase: crystallization and preliminary X-ray data.

The heat-stable aminopeptidase from Aeromonas proteolytica has been purified using two new procedures, with the aim of preparing large single crystals for X-ray analysis. In a first procedure, we tried to avoid any drastic conditions capable of inducing microheterogeneities in the protein sample. The enzyme was purified through two chromatographic steps based on hydrophobic interactions and ion exchange. In a second procedure a heat treatment of the protein to a temperature of 70 degrees C over 5 to 8 h was performed. Both procedures led to an electrophoretically homogeneous and crystallizable aminopeptidase; however, unexpectedly, the crystals obtained through the first procedure contained, in addition to the native aminopeptidase, a cleaved form of the enzyme which has been characterized. Only the native protein was present when the second procedure was used. Large crystals obtained with the native protein form, having an approximate size of 0.4 x 0.4 x 0.6 mm, produced an X-ray diffraction pattern that exhibited the symmetry associated with the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22). The unit cell parameters were a = 109.1 A and c = 97.8 A. Assuming one molecule/asymmetric unit, a value of VM = 2.6 A3/Da and an approximate solvent content of 45% could be estimated. Measurable diffraction intensities were observed at a resolution of 2.5 A.

Aeromonas↗
PubMed1992

Structural and functional relationships between aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases can be divided in two groups of equal size on the basis of differences in the structure of their active sites. The core of class I synthetases is the classical nucleotide-binding domain with its characteristic Rossmann fold. In contrast, the active site of class II synthetases is built around an antiparallel beta-sheet, to which the substrates bind. This classification, which is based on structural data (amino acid sequences and tertiary structures), can be rationalized in functional terms.

Amino Acyl-tRNA Synthetases↗
PubMed1991

Base-pairing shift in the major groove of (CA)n tracts by B-DNA crystal structures.

the crystal packing of the B-DNA dodecamer d(ACCG-GCGCCACA).d(TGTGGCGCCGGT) is characterized by the reciprocal fit of double helices with specific base-backbone interactions in the major groove. Cooling the crystals below -10 degrees C stabilizes a new conformational state with a long-range sequence-dependent one-step shift in the major-groove base pairing. The tilt of the bases leads to the disruption of the Watson-Crick pairing in the major groove and to the formation of interactions with the 5' neighbour of their complement. This alteration propagates along the helical axis over more than half a turn. As a result, the molecular structure is normal when seen from the minor groove side and mismatched in the major groove. Comparison with a parent isomorphous dodecamer structure corresponding to the codon 10-13 of the c-Ha-ras proto-oncogene show that this new structural feature is sequence dependent and clearly favoured by (CA)n tracts. As(CA)n tracts of DNA are involved both in recombination and in transcription, this new recognition pattern should be considered in the analysis of the various processes involving the reading of the genetic information.

Base Sequence↗
PubMed1991

Groove-backbone interaction in B-DNA. Implication for DNA condensation and recombination.

DNA self-fitting is revealed by the study of intermolecular contacts found in the crystal packing of a dodecamer where the helices are locked together by a reciprocal groove-backbone interaction and form a crossed structure. It is proposed that it could be a model for DNA-DNA interaction in several biological processes such as the node of supercoiled DNA and synapsis in recombination. The main topological and symmetrical features of this crossed structure are described and the symmetry-homology relationships are analyzed in the more general case of B-DNA interacting helices. Model-building of Holliday junctions with minimal change from the starting crystal coordinates of the crossed structure leads to at least three different solutions. These various models are compared from the point of view of their symmetry and topology, in the light of their branch migration and resolution properties. In addition, a model for a self-favored reciprocal unwinding mechanism based on the experimentally observed structural alterations, such as the packing-induced opening of G.C base-pairs is proposed. In this model, the phosphate groups of the invading backbone trigger the opening of the base-pairs of the other helix, by pulling cytosine or adenine bases out of the major groove after binding to their amino group.

Base Sequence↗
PubMed1991

Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes.

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N-AcPhe-tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N-AcPhe-tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319-322).

Crystallography↗
PubMed1991

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp).

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

Aspartate-tRNA Ligase↗
PubMed1990

Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.

The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction. These proteins differ widely in size and oligomeric state, and have limited sequence homology. Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP. We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS. These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS. These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.

Amino Acid Sequence↗