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D Moras

Publications and source records attributed to D Moras.

At least 19 recordsLinked to original sources
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↗
PubMed1990

Cardiotoxin VII4 from Naja mossambica mossambica. The refined crystal structure.

The crystal structure of cardiotoxin VII4 from Naja mossambica mossambica was refined to 2.5 A resolution. Fifty ordered solvent sites were localized and included in the refinement. The final R factor is 0.197 (lambda/(2sin theta) less than 5 A; F greater than 3 sigma). The three-dimensional structure is characterized by two beta-sheets. Of particular interest is the two-stranded beta-sheet in the N-terminal region. This shows a large right-handed twist and, though strongly connected to the core of the molecule, and in particular to the C-terminal end, protrudes out of the bulk of the molecule. The segment of four amino acid residues connecting the two strands of this sheet is particularly exposed. It contains an invariant proline residue that has probably an important structural role, and is completely hydrophobic. Two other conserved hydrophobic zones were identified; the largest extends over the second and third loops, on one side only of the molecule. All side-chains of invariant hydrophobic character (except proline residues) belong to one of these three zones. Also discussed are the dimeric assembly and the rather loose packing in the crystal. The three-dimensional structure is compared with that of short and long alpha-neurotoxins. Comparison with two-dimensional nuclear magnetic resonance results on the 68% homologous cardiotoxin CT X IIb shows an excellent overall agreement. A few differences are probably genuine.

Amino Acid Sequence↗
PubMed1990

The contacts of yeast tRNA(Ser) with seryl-tRNA synthetase studied by footprinting experiments.

Yeast tRNA(Ser) is a member of the class II tRNAs, whose characteristic is the presence of an extended variable loop. This additional structural feature raises questions about the recognition of these class II tRNAs by their cognate synthetase and the possibility of the involvement of the extra arm in the recognition process. A footprinting study of yeast tRNA(Ser) complexed with its cognate synthetase, yeast seryl-tRNA synthetase (an alpha 2 dimer), was undertaken. Chemical (ethylnitrosourea) and enzymatic (nucleases S1 and V1) probes were used in the experiments. A map of the contact points between the tRNA and the synthetase was established and results were analyzed with respect to a three-dimensional model of yeast tRNA(Ser). Regions in close vicinity with the synthetase are clustered on one face of tRNA. The extra arm, which is strongly protected from chemical modifications, appears as an essential part of the contact area. The anticodon triplet and a large part of the anticodon arm are, in contrast, still accessible to the probes when the complex is formed. These results are discussed in the context of the recognition of tRNAs in the aminoacylation reaction.

Amino Acyl-tRNA Synthetases↗
PubMed1990

Antithrombin III: structural and functional aspects.

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.

Amino Acid Sequence↗
PubMed1990

An attempt to unify the structure of polymerases.

With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary crystallographic data for bovine antithrombin III.

Crystals of bovine antithrombin III were obtained in the presence of metal ions with ammonium sulphate as precipitating agent. Crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with cell parameters a = b = 91.4 A, c = 383.1 A; there are two molecules per asymmetric unit. Electrophoresis experiments and amino acid sequence analysis of the N-terminal part of redissolved crystals suggest that the protein molecules are cleaved at the active site.

Animals↗