Retinoid X receptors (RXR␣, -␤, and -␥) occupy a central position in the nuclear receptor superfamily, because they form heterodimers with many other family members and hence are involved in the control of a variety of (patho)physiologic processes. Selective RXR ligands, referred to as rexinoids, are already used or are being developed for cancer therapy and have promise for the treatment of metabolic diseases. However, important side effects remain associated with existing rexinoids. Here we describe the rational design and functional characterization of a spectrum of RXR modulators ranging from partial to pure antagonists and demonstrate their utility as tools to probe the implication of RXRs in cell biological phenomena. One of these ligands renders RXR activity particularly sensitive to coactivator levels and has the potential to act as a cell-specific RXR modulator. A combination of crystallographic and fluorescence anisotropy studies reveals the molecular details accounting for the agonist-to-antagonist transition and provides direct experimental evidence for a correlation between the pharmacological activity of a ligand and its impact on the structural dynamics of the activation helix H12. Using RXR and its cognate ligands as a model system, our correlative analysis of 3D structures and dynamic data provides an original view on ligand actions and enables the establishment of mechanistic concepts, which will aid in the development of selective nuclear receptor modulators.crystal structure ͉ ligand design ͉ nuclear receptor ͉ agonist ͉ antagonist N uclear Receptor (NR)-controlled gene expression relies on a mechanism in which NRs recruit coregulators that are part of multiprotein complexes. These complexes correspond to chromatin-modifying and transcription-initiating machineries that act at target gene promoters in a precisely timed and sequential fashion (1). The binding of a ligand to the ligandbinding domain (LBD) of NRs constitutes the initial step of this regulatory process. In this context, the C-terminal helix H12 of LBDs plays a key role, because its position, which depends on the bound ligand, determines the type of coregulator recruited by the receptor (2). Structural studies have shown that in agonistbound NR LBDs, H12 adopts the so-called ''active'' or ''holo'' conformation and provides a binding surface for short NR interaction motifs of coactivators (3). In contrast, antagonists prevent H12 from adopting the holo position (4).Therapeutically, retinoid X receptor (RXR)-selective ligands, referred to as rexinoids, are used in cancer therapy, and previously uncharacterized rexinoid-based therapeutic paradigms are currently being explored. In addition, rexinoids have promise for use in the therapy of metabolic diseases (5, 6), but important side effects associated with existing compounds limit their use. Improved understanding of the biological role and the structural biology of RXR (7, 8) will allow the synthesis of selective modulators that might overcome the limitations of current drugs. Here,...

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Retinoid receptors (RARs and RXRs) are ligand-activated transcription factors that regulate the transcription of target genes by recruiting coregulator complexes at cognate promoters. To understand the effects of heterodimerization and ligand binding on coactivator recruitment, we solved the crystal structure of the complex between the RAR␤/RXR␣ ligand-binding domain heterodimer, its 9-cis retinoic acid ligand, and an LXXLLcontaining peptide (termed NR box 2) derived from the nuclear receptor interaction domain (NID) of the TRAP220 coactivator. In parallel, we measured the binding affinities of the isolated NR box 2 peptide or the fulllength NID of the coactivator SRC-1 for retinoid receptors in the presence of various types of ligands. Our correlative analysis of three-dimensional structures and fluorescence data reveals that heterodimerization does not significantly alter the structure of individual subunits or their intrinsic capacity to interact with NR box 2. Similarly, we show that the ability of a protomer to recruit NR box 2 does not vary as a function of the ligand binding status of the partner receptor. In contrast, the strength of the overall association between the heterodimer and the full-length SRC-1 NID is dictated by the combinatorial action of RAR and RXR ligands, the simultaneous presence of the two receptor agonists being required for highest binding affinity. We identified an LXXLL peptidedriven mechanism by which the concerted reorientation of three phenylalanine side chains generates an "aromatic clamp" that locks the RXR activation helix H12 in the transcriptionally active conformation. Finally, we show how variations of helix H11-ligand interactions can alter the communication pathway linking helices H11, H12, and the connecting loop L11-12 to the coactivatorbinding site. Together, our results reveal molecular and structural features that impact on the ligand-dependent interaction of the RAR/RXR heterodimer with nuclear receptor coactivators.Retinoids are the active metabolites of vitamin A that regulate complex gene networks involved in cell differentiation, proliferation, and apoptosis (1-4). Notably, they are effective inhibitors of tumor cell growth, thus supporting their actual or potential use in cancer therapy and prevention (5-7). The biological activity of all-trans and 9-cis retinoic acid (9C-RA) 1 signals are mediated by two families of nuclear receptors that act as ligand-dependent transcriptional regulators: the retinoic acid receptors (RAR␣, -␤, and -␥) and the retinoid X receptors (RXR␣, -␤, and -␥). Although RARs bind and are activated by both the 9-cis and the all-trans isomers of the retinoic acid, RXRs are exclusively activated by the 9-cis isomer (8 -10). Like other members of the nuclear receptor superfamily, retinoid receptors are modular and include an N-terminal A/B activation domain, a central DNA-binding domain (region C), a linker region D, a ligand-binding domain (region E), and a C-terminal region F of unknown structure and function. The ligand-binding domain ...

The crystal structure of the ligand-binding domain of RARb, a suspect tumour suppressor, reveals important features that distinguish it from the two other RAR isotypes. The most striking difference is an extra cavity allowing RARb to bind more bulky agonists. Accordingly, we identified a ligand that shows RARb selectivity with a 100-fold higher affinity to RARb than to a or c isotypes. The structural differences between the three RAR ligand-binding pockets revealed a rationale explaining how a single retinoid can be at the same time an RARa, c antagonist and an RARb agonist. In addition, we demonstrate how to generate an RARb antagonist by gradually modifying the bulkiness of a single substitution. Together, our results provide structural guidelines for the synthesis of RARb-selective agonists and antagonists, allowing for the first time to address pharmacologically the tumour suppressor role of RARb in vitro and in animal models.

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