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D Moras

Publications and source records attributed to D Moras.

At least 91 records · Page 5Linked to original sources
PubMed1998

The nuclear receptor ligand-binding domain: structure and function.

In the past few years our understanding of nuclear receptor action has dramatically improved as a result of the elucidation of the crystal structures of the empty (apo) ligand-binding domains of the nuclear receptor and of complexes formed by the nuclear receptor's ligand-binding domain bound to agonists and antagonists. Furthermore, the concomitant identification and functional analysis of co-regulators (transcriptional intermediary factors [TIFs], comprising co-activators and co-repressors) previously predicted from squelching studies, have deepened this understanding. Recent data have provided the structural basis for the specific recognition of ligands and the molecular mechanisms of agonism and antagonism, enabling us to gain a comprehensive view of the early steps of nuclear receptor action.

Animals↗
PubMed1998

Functional and structural analysis of the subunits of human transcription factor TFIID.

The past few years have brought many new insights concerning the structure and function of TAFII proteins. In the future, further biochemical and structural studies will no doubt lead to a greater understanding of the molecular organization of TFIID complexes. A better understanding of the function of metazoan, in particular, mammalian, TAFIIs in cell cycle progression and gene activation will, however, require the use of novel genetic techniques in addition to the biochemical analyses.

Animals↗
PubMed1997

Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP.

A connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID binds selectively to and is sequestered by cisplatin- or UV-damaged DNA, either alone or in the context of a larger protein complex including TFIIH. Computer-assisted 3D structural analysis reveals a remarkable similarity between the structure of the TATA box as found in its TBP complex and that of either platinated or UV-damaged oligonucleotides. Thus, cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP/TFIID away from its normal promoter sequence, partially explaining the phenomenon of DNA damage-induced inhibition of RNA synthesis. Consistent with an involvement of damaged DNA-specific binding of TBP in inhibiting transcription, we find that microinjection of additional TBP in living human fibroblasts alleviates the reduction in RNA synthesis after UV irradiation. Future anticancer drugs could be designed with the consideration of lesion recognition by TBP and their ability to reduce transcription.

Cisplatin↗
PubMed1997

A mutation mimicking ligand-induced conformational change yields a constitutive RXR that senses allosteric effects in heterodimers.

Mutations of a single residue in the retinoid X receptor alpha (RXRalpha) ligand-binding pocket (LBP) generate constitutive, ligand-binding-competent mutants with structural and functional characteristics similar to those of agonist-bound wild-type RXR. Modelling of the mouse RXRalphaF318A LBP suggests that, like agonist binding, the mutation disrupts a cluster of van der Waals interactions that maintains helix H11 in the apo-receptor location, thereby shifting the thermodynamic equilibrium to the holo form. Heterodimerization with some apo-receptors (retinoic acid, thyroid hormone and vitamin D3 receptors) results in 'silencing' of RXRalphaF318A constitutive activity, which, on the other hand, efficiently contributes to synergistic transactivation within NGFI-B-RXR heterodimers. RAR mutants disabled for corepressor binding and/or lacking a functional AF-2 activation domain, do not relieve RXR 'silencing'. Not only RAR agonists, but also the RAR antagonist BMS614 induce conformational changes allowing RXR to exert constitutive (RXRalphaF318A) or agonist-induced (wild-type RXR) activity in heterodimers. Interestingly, the RXRalphaF318A constitutive activity generated within heterodimers in the presence of BMS614 requires the integrity of both RXR and RAR AF-2 domains. These observations suggest that, within RXR-RAR heterodimers, RAR can adopt a structure distinct from that of the active holo-RAR, thus allowing RXR to become transcriptionally responsive to agonists.

Allosteric Regulation↗
PubMed1997

Effect of cytosine methylation on DNA-DNA recognition at CpG steps.

Although DNA methylation is a fundamental mechanism for repressing genetic activity, the influence of methyl groups on DNA conformation is found to be small. In this study, the role of cytosine methylation is analysed in the context of DNA condensation by examining its influence on DNA-DNA recognition processes. Previously CpG sites were found to act as sequence determinants for the close and specific self-fit of B-DNA helices into cross-overs. In the present study, the crystal structure of the B-DNA dodecamer d(ACCGCCGGCGCC) methylated at its central CpG sequence shows that the methyl groups do not interfere with DNA self-fitting. In contrast, the two methyl groups form a clamp, which traps the incoming phosphate in the groove-backbone interaction. This geometry allows the formation of two new C-H...O hydrogen bonds between the methyl groups and the anionic oxygen atoms of the phosphate, which may further stabilize the interaction. This finding relates cytosine methylation to the formation of higher-order DNA structures and could provide new insights for understanding the mode of action of DNA methylation in genetic inactivation.

Crystallography, X-Ray↗
PubMed1997

The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase.

The crystal structure of an enzyme-substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme-product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between beta- and gamma-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

Acylation↗
PubMed1997

The structure of Staphylococcus aureus epidermolytic toxin A, an atypic serine protease, at 1.7 A resolution.

BACKGROUND: Staphylococcal epidermolytic toxins A and B (ETA and ETB) are responsible for the staphylococcal scalded skin syndrome of newborn and young infants; this condition can appear just a few hours after birth. These toxins cause the disorganization and disruption of the region between the stratum spinosum and the stratum granulosum--two of the three cellular layers constituting the epidermis. The physiological substrate of ETA is not known and, consequently, its mode of action in vivo remains an unanswered question. Determination of the structure of ETA and its comparison with other serine proteases may reveal insights into ETA's catalytic mechanism. RESULTS: The crystal structure of staphylococcal ETA has been determined by multiple isomorphous replacement and refined at 1.7 A resolution with a crystallographic R factor of 0.184. The structure of ETA reveals it to be a new and unique member of the trypsin-like serine protease family. In contrast to other serine protease folds, ETA can be characterized by ETA-specific surface loops, a lack of cysteine bridges, an oxyanion hole which is not preformed, an S1 specific pocket designed for a negatively charged amino acid and an ETA-specific specific N-terminal helix which is shown to be crucial for substrate hydrolysis. CONCLUSIONS: Despite very low sequence homology between ETA and other trypsin-like serine proteases, the ETA crystal structure, together with biochemical data and site-directed mutagenesis studies, strongly confirms the classification of ETA in the Glu-endopeptidase family. Direct links can be made between the protease architecture of ETA and its biological activity.

Amino Acid Sequence↗
PubMed1997

A 'specificity' pocket inferred from the crystal structures of the complexes of aldose reductase with the pharmaceutically important inhibitors tolrestat and sorbinil.

BACKGROUND: Aldose reductase (AR) is an NADPH-dependent enzyme implicated in long-term diabetic complications. Buried at the bottom of a deep hydrophobic cleft, the NADPH coenzyme is surrounded by the conserved hydrophilic residues of the AR active site. The existence of an anionic binding site near the NADP+ has been determined from the structures of the complexes of AR with citrate, cacodylate and glucose-6-phosphate. The inhibitor zopolrestat binds to this anionic site, and in the hydrophobic cleft, after a change of conformation which opens a 'specificity' pocket. RESULTS: The crystal structures of the porcine AR holoenzyme and its complexes with the inhibitors tolrestat and sorbinil have been solved; these structures are important as tolrestat and sorbinil are, pharmaceutically, the most well-studied AR inhibitors. The active site of the holoenzyme was analyzed, and binding of the inhibitors was found to involve two contact zones in the active site: first, a recognition region for hydrogen-bond acceptors near the coenzyme, with three centers, including the anionic site; and second, a hydrophobic contact zone in the active-site cleft, which in the case of tolrestat includes the specificity pocket. The conformational change leading to the opening of the specificity pocket upon tolrestat binding is different to the one seen upon zopolrestat binding; this pocket binds inhibitors that are more effective against AR than against aldehyde reductase. CONCLUSIONS: The active site of AR adapts itself to bind tightly to different inhibitors; this happens both upon binding to the inhibitor's hydrophilic heads, and at the hydrophobic and specificity pockets of AR, which can change their shape through different conformational changes of the same residues. This flexibility could explain the large variety of possible substrates of AR.

Aldehyde Reductase↗
PubMed1997

The structure of cyclin H: common mode of kinase activation and specific features.

The crystal structure of human cyclin H refined at 2.6 A resolution is compared with that of cyclin A. The core of the molecule consists of two repeats containing five helices each and forming the canonical cyclin fold also observed in TFIIB. One hundred and thirty-two out of the 217 C alpha atoms from the cyclin fold can be superposed with a root-mean-square difference of 1.8 A. The structural homology is even higher for the residues at the interface with the kinase, which is of functional significance, as shown by our observation that cyclin H binds to cyclin-dependent kinase 2 (cdk2) and that cyclin A is able to activate cdk7 in the presence of MAT1. Based on this superposition, a new signature sequence for cyclins was found. The specificity of the cyclin H molecule is provided mainly by two long helices which extend the cyclin fold at its N- and C-termini and pack together against the first repeat on the side opposite to the kinase. Deletion mutants show that the terminal helices are required for a functionally active cyclin H.

Amino Acid Sequence↗
PubMed1997

Purification of the human RARgamma ligand-binding domain and crystallization of its complex with all-trans retinoic acid.

A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity. The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM. Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method. They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca. 33%, and a Vm value of approximately 2 A3/dalton.

Binding Sites↗
PubMed1997

Structural and functional considerations of the aminoacylation reaction.

Aminoacyl-tRNA synthetases (aaRS) bind their substrates-ATP, amino acids and tRNA- and stabilize putative transition states in the aminoacylation reaction. Here, we discuss the common and distinguishing structural and functional themes of the 20 known aaRS, which can be divided into two main classes (I and II) and into further subgroups on this basis.

Adenosine Triphosphate↗
PubMed1997

Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant.

The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.

Aspartate-tRNA Ligase↗
PubMed1997

A transcriptional silencing domain in DAX-1 whose mutation causes adrenal hypoplasia congenita.

The DAX-1 gene encodes an unusual member of the nuclear hormone receptor superfamily. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. We have shown that DAX-1 binds to hairpin secondary structures and blocks steroidogenesis in adrenal cells via transcriptional repression of the steroidogenic acute regulatory protein (StAR) promoter. Here we have investigated the molecular mechanism of DAX-1-mediated repression. We show that the DAX-1 C terminus contains a potent transcriptional silencing activity, which can be transferred to a heterologous DNA-binding domain. Deletion analysis and modeling of DAX-1 structure identify two cooperating domains required for the silencing function, one located within helix H3 and the other within H12. The silencing function is cell- and promoter-specific. Strikingly, two point mutations (R267P and deltaV269) found in adrenal hypoplasia patients impair silencing. These findings suggest that transcriptional silencing by DAX-1 plays a critical role in the pathogenesis of adrenal hypoplasia congenita.

Adrenal Glands↗
PubMed1996

Genomic organization of the rat aspartyl-tRNA synthetase gene family: a single active gene and several retropseudogenes.

The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8-16, encoding the nt-binding domain of the synthetase, are clustered in the 3'-region of the gene, whereas exons 3, 4, and 5, encoding the anticodon-binding domain are separated by large introns (up to 15 kb) containing LINE sequences. One of the pseudogenes, psi DRS1, has a nt sequence 93% identical to that of the complete cDNA sequence of rat AspRS but several stop codons interrupt the coding sequence, thus identifying psi DRS1 to an inactive processed pseudogene. Two repetitive elements from the LINE family are inserted into psi DRS1. Calculation of nt substitution rates suggests that psi DRS1 sequences arose approximately 27 Myr ago. The other pseudogene, psi DRS2, should be more ancient. Taken together, these results clearly demonstrate that the AspRS gene family is composed of only one active gene. The availability of the gene structure of AspRS could help to clarify molecular evolution of class II aaRS.

Amino Acid Sequence↗
PubMed1996

The crystal structure of human cyclin H.

The crystal structure of human cyclin H has been solved at 2.6 A resolution by the MIR method and refined to an R-factor of 23.1%. The core of the molecule consists of two helical repeats adopting the canonical cyclin fold already observed in the structures of cyclin A [Brown et al. (1995) Structure 3, 1235-1247; Jeffrey et al. (1995) Nature 376, 313-320; Russo et al. (1996) Nature 382, 325-331] and TFIIB [Nikoilov et al. (1995) Nature 377, 119-128]. The N-terminal and C-terminal residues form a new domain built on two long helices interacting essentially with the first repeat of the molecule.

Crystallization↗