Detection of nucleic acid-nuclear hormone receptor complexes with mass spectrometry

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR·RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR·RXR·DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RA

Versatile copurification procedure for rapid isolation of homogeneous RAR-RXR heterodimers

International audienceRAR-RXR heterodimeric complexes (RARalphaDeltaAB-RXRalphaDeltaAB) bound to cognate DR5 DNA response elements were purified to apparent structural and functional homogeneity using a novel versatile immobilized metal affinity chromatography (IMAC) copurification procedure. Dynamic light scattering studies indicated that the complexes were more than 85% monodisperse. By electron microscopy the negatively stained RAR-RXR-DNA complexes appeared homogeneous and corresponded to a dimeric arrangement of the molecules. Using heterodimers purified according to this procedure we demonstrate ligand binding of RXR in the context of the RAR-RXR heterodimer-DNA complex. The present copurification procedure is rapid and has yielded high quality heterodimer-DNA complexes suitable for both structural and biochemical studies

Advances in vitamin D receptor function and evolution based on the 3D structure of the lamprey ligand binding domain

International audience1α,25-dihydroxyvitamin D 3 (1,25D 3) regulates many physiological processes in vertebrates by binding to the Vitamin D Receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand binding domain complex from the basal vertebrate Petromyzon marinus, sea lamprey (lVDR). Comparison of 3D crystal structure of lVDR-1,25D 3 complex with higher vertebrates VDR-1,25D 3 structures suggest that 1,25D 3 binds to lVDR similarly to human VDR (hVDR), but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Further, residue co-evolution analysis among vertebrates VDR identifies amino-acid positions in H9 and the large insertion domain (iD) VDR LBD specific

Monitoring of the retinoic acid receptor–retinoid X receptor dimerization upon DNA binding by native mass spectrometry

International audienceIdentifying protein-DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor-retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR-RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR-RXR heterodimerization

Detection of Nucleic Acid-Nuclear Hormone Receptor Complexes with Mass Spectrometry

ISSN:1879-1123ISSN:1044-030

4-Hydroxy-1α,25-Dihydroxyvitamin D₃: Synthesis and Structure–Function Study

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure–activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4β,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4β,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound

RARβ ligand-binding domain bound to an SRC-1 co-activator peptide: purification, crystallization and preliminary X-ray diffraction analysis

International audienceRetinoids have demonstrated therapeutic efficacy in the treatment of acute promyelocytic leukaemia and in the chemoprevention of a large number of cancers. As the cellular signalling pathway of retinoids can be transduced by the three retinoic acid receptor (RAR) isotypes alpha, beta and gamma, the side effects of these treatments induced efforts to generate isotype-selective ligands. Despite knowledge of the crystal structures of RARalpha and RARgamma ligand-binding domains (LBDs), the rational design of such ligands has been hampered by the absence of RARbeta LBD structural data. Here, a strategy used to express a large-scale soluble fraction of the human RARbeta LBD suitable for biophysical analysis is reported, as well as a procedure for crystallizing it bound to a synthetic retinoid (TTNPB) with or without a co-activator peptide (SRC-1). Preliminary X-ray analysis revealed that both complexes crystallized in the orthorhombic space group P2(1)2(1)2(1). The unit-cell parameters are a = 47.81, b = 58.52, c = 92.83 A for the TTNPB-hRARbeta LBD crystal and a = 58.14, b = 84.07, c = 102.37 A when the SRC-1 peptide is also bound

Design, synthesis and evaluation of side-chain hydroxylated derivatives of lithocholic acid as potent agonists of the vitamin D receptor (VDR)

International audienceA high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest

Modulation of RXR-DNA complex assembly by DNA context

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Design, Synthesis, and Biological Evaluation of New Type of Gemini Analogues with a Cyclopropane Moiety in Their Side Chain

International audienceWe synthesized two new gemini analogues UG-480 and UG-481 that incorporate a modified longer side chain containing a cyclopropane group. The evaluation of the bioactivities of the two gemini analogues indicated that the 17,20 threo (20S) compound, UG-480, is the most active one and as active as 1,25(OH)2D3. Docking and MD data showed that the compounds bind efficiently to VDR with UG-480 forming an energetically more favorable interaction with His397. Structural analysis indicated that whereas the UG-480 compound efficiently stabilizes the active VDR conformation, it induces conformational changes in H6-H7 VDR region that are greater than those induced by the parental Gemini and that this is due to the occupancy of the secondary channel by its modified side chain