Structural and biochemical basis for selective repression of the orphan nuclear receptor liver receptor homolog 1 by small heterodimer partner

Abstract

The functional interaction between the orphan nuclear receptors small heterodimer partner (SHP) and liver receptor homolog 1 (LRH-1), where SHP binds to LRH-1 and represses its constitutive transcriptional activity, is crucial for regulating genes involved in cholesterol homeostasis. Here, we report structural and biochemical analyses of the LRH-1͞SHP interaction. The crystal structure and modeling studies of the LRH-1 ligand-binding domain bound to either of the two LXXLL-related motifs of SHP show that the receptor undergoes conformational changes to accommodate the SHP docking and reveal key residues that determine the potency and selectivity of SHP binding. Through a combination of mutagenesis and binding studies, we demonstrate that only the second SHP LXXLL motif is required for repressing LRH-1, and this motif displays a strong preference for binding to LRH-1 over the closely related receptor steroidogeneic factor 1 (SF-1). Structural comparisons indicate that this binding selectivity is determined by residues flanking the core LXXLL motifs. These results establish a structural model for understanding how SHP interacts with LRH-1 to regulate cholesterol homeostasis and provide new insights into how nuclear receptor͞coregulator selectivity is achieved.steroidogeneic factor 1 ͉ bile acids ͉ coactivators ͉ corepressors L iver receptor homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that activates an array of genes responsible for development of endodermal organs such as liver, intestine, and pancreas (reviewed in ref. 1). LRH-1 also plays a central role in lipid homeostasis by regulating genes involved in bile acid synthesis, reverse cholesterol transport, and metabolism of lipoprotein complexes (2-6). In the nuclear receptor superfamily, LRH-1 is most homologous to steroidogeneic factor 1 (SF-1, NR5A1), which is essential for sex differentiation and development of adrenals and gonads (7,8). LRH-1 and SF-1 share a highly conserved DNA-binding domain (DBD, Ͼ90% identity) and a moderately conserved ligand-binding domain (LBD, 56% identity). In contrast to most other nuclear receptors that function as dimers, LRH-1 and SF-1 bind with high affinity as monomers to a conserved consensus DNA site found in the promoters of target genes (9). The high degree of similarity in the DBDs of LRH-1 and SF-1 suggests that their different biological activities are contributed in part by distinct structural features in their LBDs, which recruit specific cofactors to regulate transcription.LRH-1 seems to be a constitutively active transcription factor in that it activates many reporters in the absence of any exogenous ligands. The crystal structure of the mouse LRH-1 LBD reveals a sandwich fold of four layers of helices instead of the three layers observed for many other receptors (10). Despite the absence of any ligand in the large ligand-binding pocket, the C-terminal activation helix (AF-2) of LRH-1 is packed in an active conformation. The constitutive LRH-1 activity is proposed to be the result of the stabilization o...

The structure of corepressor Dax-1 bound to its target nuclear receptor LRH-1

“…The GST and the GST protein fusions with LRH-1 and SF-1 LBDs were expressed in Escherichia coli and immobilized on glutathioneagarose beads (Sigma). 35 S-radiolabelled full-length mDax-1 was prepared in vitro in a reticulocyte lysate using the TNT T7 quick-coupled transcriptiontranslation system (Promega) according to the manufacturer's protocol. The immobilized GST proteins with specifically bound mDax-1 were eluted, resolved by SDS-PAGE, and visualized by autoradiography.…”

“…To evaluate whether Dax-1 can bind to LRH-1 in vitro, we performed the standard GST pull-down assay using bacterially expressed and purified GST-LRH-1 LBD fusion protein and in vitro transcribed and translated 35 S-labeled full-length Dax-1. The results of this experiment show that Dax-1 interacted with LRH-1 LBD in the absence of any added hormones or coregulatory proteins.…”

Structural insights into gene repression by the orphan nuclear receptor SHP

“…The binding of the cofactor peptides to SHP was determined by AlphaScreen luminescence proximity assays as described (8,18,41). Reaction mixtures consisted of 50 nM HisMBP fusion proteins, 200 nM biotinylated peptides/proteins, 10 μg/mL nickel chelate-coated acceptor beads (PerkinElmer Life Sciences), and 10 μg/mL streptavidin-coated donor beads (PerkinElmer Life Sciences) in a buffer containing 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.4, 50 mM NaF, 50 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 0.1 mg/mL BSA.…”

“…Inclusion of short peptides containing coactivator LXXLL motifs or corepressor LXXXLXXX(L/I) motifs has facilitated the crystallization and diffraction of a number of nuclear receptor LBDs (4,8,18). We thus examined the interaction of HisMBP-SHPLBD (X6, Fig.…”

“…In the first step, SHP uses an internal LXXLL motif to bind to the AF-2 site of its target nuclear receptors, the same site that is required for the binding of coactivators (18). The LXXLL motif of SHP has a relatively high binding affinity for a number of nuclear receptors (18), and SHP binding to the AF-2 site of these nuclear receptors causes the release of coactivators, thus decreasing gene activation. In the second step, SHP actively recruits corepressors to inhibit gene transcription.…”