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

Structural Basis for alpha-Helix Mimicry and Inhibition of Protein-Protein Interactions with Oligourea Foldamers

Efficient optimization of a peptide lead into a drug candidate frequently needs further transformation to augment properties such as bioavailability. Among the different options, foldamers, which are sequence-based oligomers with precise folded conformation, have emerged as a promising technology. We introduce oligourea foldamers to reduce the peptide character of inhibitors of protein-protein interactions (PPI). However, the precise design of such mimics is currently limited by the lack of structural information on how these foldamers adapt to protein surfaces. We report a collection of X-ray structures of peptide-oligourea hybrids in complex with ubiquitin ligase MDM2 and vitamin D receptor and show how such hybrid oligomers can be designed to bind with high affinity to protein targets. This work should enable the generation of more effective foldamer-based disruptors of PPIs in the context of peptide lead optimization

PLoS Genet

The retinoid X receptors (RXRs) are ligand-activated transcription factors which heterodimerize with a number of nuclear hormone receptors, thereby controlling a variety of (patho)-physiological processes. Although synthetic RXR ligands are developed for the treatment of various diseases, endogenous ligand(s) for these receptors have not been conclusively identified. We show here that mice lacking cellular retinol binding protein (Rbp1-/-) display memory deficits reflecting compromised RXR signaling. Using HPLC-MS and chemical synthesis we identified in Rbp1-/- mice reduced levels of 9-cis-13,14-dihydroretinoic acid (9CDHRA), which acts as an RXR ligand since it binds and transactivates RXR in various assays. 9CDHRA rescues the Rbp1-/- phenotype similarly to a synthetic RXR ligand and displays similar transcriptional activity in cultured human dendritic cells. High endogenous levels of 9CDHRA in mice indicate physiological relevance of these data and that 9CDHRA acts as an endogenous RXR ligand

1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

Background: The 1 alpha,25-dihydroxy-3-epi-vitamin-D(3) (1 alpha,25(OH)(2)-3-epi-D(3)), a natural metabolite of the seco-steroid vitamin D(3), exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1 alpha,25(OH)(2)-3-epi-D(3) is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1 alpha,25(OH)(2)D(3). To further unveil the structural mechanism and structure-activity relationships of 1 alpha,25(OH)(2)-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD). Methodology/Principal Findings: In the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1 alpha,25(OH)(2)D(3). We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1 alpha,25(OH)(2)-3-epi-D(3) in primary human keratinocytes and biochemical properties are comparable to 1 alpha,25(OH)(2)D(3). Conclusions/Significance: The physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1 alpha,25(OH)(2)D(3) lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1 alpha,25(OH)(2)D(3)

Vitamin D and Its Receptor from a Structural Perspective

The activities of 1α,25-dihydroxyvitamin D3, 1,25D3, are mediated via its binding to the vitamin D receptor (VDR), a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily. Numerous studies have demonstrated the important role of 1,25D3 and VDR signaling in various biological processes and associated pathologies. A wealth of information about ligand recognition and mechanism of action by structural analysis of the VDR complexes is also available. The methods used in these structural studies were mainly X-ray crystallography complemented by NMR, cryo-electron microscopy and structural mass spectrometry. This review aims to provide an overview of the current knowledge of VDR structures and also to explore the recent progress in understanding the complex mechanism of action of 1,25D3 from a structural perspective

Vitamin D and Its Receptor from a Structural Perspective

International audienceThe activities of 1α,25-dihydroxyvitamin D3, 1,25D3, are mediated via its binding to the vitamin D receptor (VDR), a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily. Numerous studies have demonstrated the important role of 1,25D3 and VDR signaling in various biological processes and associated pathologies. A wealth of information about ligand recognition and mechanism of action by structural analysis of the VDR complexes is also available. The methods used in these structural studies were mainly X-ray crystallography complemented by NMR, cryo-electron microscopy and structural mass spectrometry. This review aims to provide an overview of the current knowledge of VDR structures and also to explore the recent progress in understanding the complex mechanism of action of 1,25D3 from a structural perspective

Ligand binding domain of vitamin D receptors.

International audienceThe vitamin D receptor, a member of the nuclear receptor subgroup NR1I, is regulated by 1alpha,25(OH)2D3 to control calcium metabolism, cell proliferation and differentiation and immunomodulation. The therapeutic applications of vitamin D metabolites are wide. To develop efficient therapy, the elucidation of the structure-function relationships of VDR and its ligands are essential. In this review we will focus on the current structural understanding of the interactions of ligands in the ligand binding pocket of the VDR. These structures revealed the mutual adaptability of the ligands and the protein. In silico modeling has further revealed a possible new pocket in the VDR LBD responsible of the non-genomic action mediated by VDR. With the availability of all these structural information on VDR LBD, new ligands that are more selective, such as non-steroidal ligands, could be designed by taking into account the flexibility of some VDR regions. Tissue selectivity may also be achieved by developing ligands that specifically activate the non-genomic pathway