Characterization of the Interaction between Retinoic Acid Receptor/Retinoid X Receptor (RAR/RXR) Heterodimers and Transcriptional Coactivators through Structural and Fluorescence Anisotropy Studies

Abstract

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 molecular basis of the interactions between synthetic retinoic acid analogues and the retinoic acid receptors

“…42 The docked structure is positioned more towards the top of the pocket, but the two structures both adopt conformation 1 and the overall positioning is very similar, thus providing further confidence in the docking protocol (the binding pose of 9CRA in RARβ as calculated by docking was also compared to the literature crystal structure PDB: 1XDK, 15 showing equally good agreement, see ESI). When the highest scoring solution of ATRA in RARγ was superimposed, the two poses were very similar, with the exception that the β-ionone ring adopted opposite orientations, and the methyl at the carboxylate end was found to be pointed downwards, rather than upwards.…”

“…It is important to note that the carboxylic acid moiety was considered as a carboxylate, rather than protonated, as this is more realistic with respect to physiological pH, and to the likelihood that the negative charge is stabilised by the closely positioned polar residues in the binding pocket. 15,32 ChemScore was chosen as the most appropriate target function in the genetic algorithm to balance between computational speed and the reliability of the GOLD predictions for the possible conformations of the different retinoids binding to RARs. 33 The genetic algorithm parameters were based on previous examples of docking hydrophobic ligands: population size 100; number of islands 5; niche size 2; selection pressure 1.1; migrate 2; and number of operators 100,000.…”

“…13,14 The retinoid-bound conformation provides a stable platform for the recruitment and binding of coactivator peptides and dimerization to the related retinoid-X-receptors (RXR). [15][16][17] Sequence alignment of RARα, β and γ shows that only three residues in the binding pocket are variable, i.e. RARγ exhibits Ala234 (Ser232 in α and Ala225 in β), Met272 (Ile270 in α and Ile263 in β) and Ala397 (Val395 in α and Val388 in β).…”

Retinoid X receptors: common heterodimerization partners with distinct functions

“…The exact physical basis for this functional synergy is unknown, however, mutagenesis studies and structural studies suggest that one coactivator molecule is recruited per heterodimer, with a single LXXLL motif being anchored to each partner 83 . Intriguingly enough, the structural studies dismissed the possibility of allosteric phenomenons between heterodimeric partners, at least for RAR-RXR heterodimers 84 . In contrast, mutagenesis studies of the RXR-LXR complex spoke in favor of allosteric interactions between the two components of the dimer 85 , a difference which could explain the permissive nature of the LXR-RXR dimer.…”

Effect of heterodimer partner RXRα on PPARγ activation function-2 helix in solution

“…controversial, as reported in the crystallographic studies on the estrogen receptor LBD homodimer [2] and retinoic acid receptor-RXR LBD heterodimer [3].…”