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

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

International audienc

Design, Synthesis, Biological Activity, and Structural Analysis of Novel Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D 3

International audienceThe toxic calcemic effects of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3, 1,25-dihydroxycholecalciferol) in the treatment of hyperproliferative diseases demand the development of highly active and noncalcemic vitamin D analogues. We report the development of two highly active and noncalcemic analogues of 1,25D3 that lack the C-ring and possess an mphenylene ring that replaces the natural D-ring. The new analogues (3a, 3b) are characterized by an additional six-carbon hydroxylated side chain attached either to the aromatic nucleus or to the triene system. Both compounds were synthesized by the Pd-catalyzed tandem cyclization/cross coupling approach starting from alkyne 6 and diphenol 8. Key steps include a stereoselective Cu-assisted addition of a Grignard reagent to an aromatic alkyne and a Takai olefination of an aromatic aldehyde. The new compounds are noncalcemic and show transcriptional and antiproliferative activities similar to 1,25D3. Structural analysis revealed that they induce a large conformational rearrangement of the vitamin D receptor around helix 6

Structural and Functional Insights into the Ligand-binding Domain of a Nonduplicated Retinoid X Nuclear Receptor from the Invertebrate Chordate Amphioxus.

International audienceRetinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR ligand-binding domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs