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

Vivian Pogenberg,Jean-François Guichou,Valérie Vivat-Hannah,Sabrina Kammerer,Efrén Pérez,Pierre Germain,Angel R De Lera,Hinrich Gronemeyer,Catherine A Royer,William Bourguet

doi:10.1074/jbc.m409302200

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 RARbeta/RXRalpha ligand-binding domain heterodimer, its 9-cis retinoic acid ligand, and an LXXLL-containing 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 full-length 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 peptide-driven 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 coactivator-binding 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.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The strength of the overall association between the heterodimer and the full-length SRC-1 nuclear receptor interaction domain (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
ApoRAR is able to synergize with CD3254 (Fig. 3C; apo/CD3254) to strongly enhance the overall binding of SRC-1570–780 as compared with SRC-1 NR box 2 or to the heterodimer bound to the RAR antagonist BMS614 (BMS614/ CD3254), the similar recruitment of SRC-1 NR box 2 and SRC1570–780 by the AM80/apo or AM80/UVI3003 complexes clearly suggests that the unliganded RXR only marginally interacts with LXXLL motifs

Summary

EXPERIMENTAL PROCEDURES

Peptides and Ligands—The human SRC-1570–780 coactivator fragment was purified and labeled with Alexa Fluor 488 as described [24]. Fluorescent peptide (fluorescein-␤A-686RHKILHRLLQEGS698) corresponding to the NR box 2-binding motif of SRC-1 was purchased from Neosystem (Strasbourg, France). Unlabeled peptides corresponding to NR box 1 (629TSHKLVQLLTTTA641), NR box 2 (686RHKILHRLLQEGS698), NR box 3 (745DHQLLRYLLDKDE757) of SRC-1, and NR box 2 of human TRAP220 coactivator (641NHPMLMNLLKDNPA654) were prepared on a Pioneer peptide synthesis system from Applied Biosystems (Solid Phase Peptide Synthesis) using 2-(1H-benzotriazole1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) activation for Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry; coupling was carried out with a 4-fold excess of activated amino acid for a minimum of 30 min. Peptides were treated with a mixture of 88% trifluoroacetic acid, 5% water, 5% phenol, and 2% triisopropylsilane for cleavage from the resin and side-chain deprotection and purified on analytical high pressure liquid chromatography using Waters C-18 columns (Waters).

TABLE I Crystallographic analysis

RESULTS AND DISCUSSION

Dissociation constants