Biomedical subjects

Dino Moras

Publications and source records attributed to Dino Moras.

60 records · Page 4Linked to original sources

PubMed2002

Expression of FLAG fusion proteins in insect cells: application to the multi-subunit transcription/DNA repair factor TFIIH.

The multi-subunit transcription/DNA repair factor TFIIH was used as a model system to show that the expression of FLAG fusion proteins in insect cells constitutes a versatile tool for both structural and functional investigations. In the present study, we have constructed recombinant baculoviruses expressing the four core TFIIH subunits fused at their N-terminus to the FLAG peptide. Using these recombinant viruses we have established protocols based on anti-FLAG immunoaffinity chromatography that allow the systematic analysis of pairwise interaction within multiprotein complexes and have developed a double tag strategy (FLAG and hexahistidine tags) for the identification and purification of stable TFIIH subcomplexes. A simple purification procedure was developed that leads to the isolation of recombinant TFIIH containing the full set of subunits. The purified recombinant TFIIH was shown to be active in a transcription assay and to be structurally homologous to the endogenous complex by electron microscopy and image analysis.

Animals↗

PubMed2002

C-H...O hydrogen bonds in the nuclear receptor RARgamma--a potential tool for drug selectivity.

Hydrogen bonds between polarized atoms play a crucial role in protein interactions and are often used in drug design, which usually neglects the potential of C-H...O hydrogen bonds. The 1.4 A resolution crystal structure of the ligand binding domain of the retinoic acid receptor RARgamma complexed with the retinoid SR11254 reveals several types of C-H...O hydrogen bonds. A striking example is the hydroxyl group of the ligand that acts as an H bond donor and acceptor, leading to a synergy between classical and C-H...O hydrogen bonds. This interaction introduces both specificity and affinity within the hydrophobic ligand pocket. The similarity of intraprotein and protein-ligand C-H...O interactions suggests that such bonds should be considered in rational drug design approaches.

Carbon↗

PubMed2002

Structural and functional evidence for ligand-independent transcriptional activation by the estrogen-related receptor 3.

The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor 3 (ERR3) complexed with a steroid receptor coactivator-1 (SRC-1) peptide reveals a transcriptionally active conformation in absence of any ligand. The structure explains why estradiol does not bind ERRs with significant affinity. Docking of the previously reported ERR antagonists, diethylstilbestrol and 4-hydroxytamoxifen, requires structural rearrangements enlarging the ligand binding pocket that can only be accommodated with an antagonist LBD conformation. Mutant receptors in which the ligand binding cavity is filled up by bulkier side chains still interact with SRC-1 in vitro and are transcriptionally active in vivo, but are no longer efficiently inactivated by diethylstilbestrol or 4-hydroxytamoxifen. These results provide structural and functional evidence for ligand-independent transcriptional activation by ERR3.

Amino Acid Sequence↗

PubMed2002

Structural basis of translational control by Escherichia coli threonyl tRNA synthetase.

Escherichia coli threonyl-tRNA synthetase (ThrRS) represses the translation of its own messenger RNA by binding to an operator located upstream of the initiation codon. The crystal structure of the complex between the core of ThrRS and the essential domain of the operator shows that the mRNA uses the recognition mode of the tRNA anticodon loop to initiate binding. The final positioning of the operator, upon which the control mechanism is based, relies on a characteristic RNA motif adapted to the enzyme surface. The finding of other thrS operators that have this conserved motif leads to a generalization of this regulatory mechanism to a subset of Gram-negative bacteria.

Anticodon↗

PubMed2002

Molecular recognition of agonist ligands by RXRs.

The nuclear receptor RXR is an obligate partner in many signal transduction pathways. We report the high-resolution structures of two complexes of the human RXRalpha ligand-binding domain specifically bound to two different and chemically unrelated agonist compounds: docosa hexaenoic acid, a natural derivative of eicosanoic acid, present in mammalian cells and recently identified as a potential endogenous RXR ligand in the mouse brain, and the synthetic ligand BMS 649. In both structures the RXR-ligand-binding domain forms homodimers and exhibits the active conformation previously observed with 9-cis-RA. Analysis of the differences in ligand-protein contacts (predominantly van der Waals forces) and binding cavity geometries and volumes for the several agonist-bound RXR structures clarifies the structural features important for ligand recognition. The L-shaped ligand-binding pocket adapts to the diverse ligands, especially at the level of residue N306, which might thus constitute a new target for drug-design. Despite its highest affinity 9-cis-RA displays the lowest number of ligand-protein contacts. These structural results support the idea that docosa hexaenoic acid and related fatty acids could be natural agonists of RXRs and question the real nature of the endogenous ligand(s) in mammalian cells.

Alitretinoin↗

PubMed2001

NF-Y recruitment of TFIID, multiple interactions with histone fold TAF(II)s.

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAF(II)s)-containing histone fold domains in protein-protein interactions and transfections. hTAF(II)20, hTAF(II)28, and hTAF(II)18-hTAF(II)28 bind to the NF-Y B-NF-YC histone fold dimer; hTAF(II)80 and hTAF(II)31-hTAF(II)80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold alpha2 helix of hTAF(II)80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAF(II)80 delta. Expression of hTAF(II)28 and hTAF(II)18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAF(II)20 and hTAF(II)135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAF(II)s, potentially accommodating different core promoter architectures.

Animals↗