Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket.

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.

Silencing LRH-1 in colon cancer cell lines impairs proliferation and alters gene expression programs.

Colorectal cancers (CRCs) account for nearly 10% of all cancer deaths in industrialized countries. Recent evidence points to a central role for the nuclear receptor liver receptor homolog-1 (LRH-1) in intestinal tumorigenesis. Interaction of LRH-1 with the Wnt/ß-catenin pathway, highly active in a critical subpopulation of CRC cells, underscores the importance of elucidating LRH-1's role in this disease. Reduction of LRH-1 diminishes tumor burden in murine models of CRC; however, it is not known whether LRH-1 is required for tumorigenesis, for proliferation, or for both. In this work, we address this question through shRNA-mediated silencing of LRH-1 in established CRC cell lines. LRH-1 mRNA knockdown results in significantly impaired proliferation in a cell line highly expressing the receptor and more modest impairment in a cell line with moderate LRH-1 expression. Cell-cycle analysis shows prolongation of G0/G1 with LRH-1 silencing, consistent with LRH-1 cell-cycle influences in other tissues. Cluster analysis of microarray gene expression demonstrates significant genome wide alterations with major effects in cell-cycle regulation, signal transduction, bile acid and cholesterol metabolism, and control of apoptosis. This study demonstrates a critical proproliferative role for LRH-1 in established colon cancer cell lines. LRH-1 exerts its effects via multiple signaling networks. Our results suggest that selected CRC patients could benefit from LRH-1 inhibitors.

The signaling phospholipid PIP3 creates a new interaction surface on the nuclear receptor SF-1.

The signaling phosphatidylinositol lipids PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind nuclear receptor 5A family (NR5As), but their regulatory mechanisms remain unknown. Here, the crystal structures of human NR5A1 (steroidogenic factor-1, SF-1) ligand binding domain (LBD) bound to PIP2 and PIP3 show the lipid hydrophobic tails sequestered in the hormone pocket, as predicted. However, unlike classic nuclear receptor hormones, the phosphoinositide head groups are fully solvent-exposed and complete the LBD fold by organizing the receptor architecture at the hormone pocket entrance. The highest affinity phosphoinositide ligand PIP3 stabilizes the coactivator binding groove and increases coactivator peptide recruitment. This receptor-ligand topology defines a previously unidentified regulatory protein-lipid surface on SF-1 with the phosphoinositide head group at its nexus and poised to interact with other proteins. This surface on SF-1 coincides with the predicted binding site of the corepressor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region on chromosome X), and importantly harbors missense mutations associated with human endocrine disorders. Our data provide the structural basis for this poorly understood cluster of human SF-1 mutations and demonstrates how signaling phosphoinositides function as regulatory ligands for NR5As.

Structure-based discovery of antagonists of nuclear receptor LRH-1.

Liver receptor homolog 1 (nuclear receptor LRH-1, NR5A2) is an essential regulator of gene transcription, critical for maintenance of cell pluripotency in early development and imperative for the proper functions of the liver, pancreas, and intestines during the adult life. Although physiological hormones of LRH-1 have not yet been identified, crystallographic and biochemical studies demonstrated that LRH-1 could bind regulatory ligands and suggested phosphatidylinositols as potential hormone candidates for this receptor. No synthetic antagonists of LRH-1 are known to date. Here, we identify the first small molecule antagonists of LRH-1 activity. Our search for LRH-1 modulators was empowered by screening of 5.2 million commercially available compounds via molecular docking followed by verification of the top-ranked molecules using in vitro direct binding and transcriptional assays. Experimental evaluation of the predicted ligands identified two compounds that inhibit the transcriptional activity of LRH-1 and diminish the expression of the receptor's target genes. Among the affected transcriptional targets are co-repressor SHP (small heterodimer partner) as well as cyclin E1 (CCNE1) and G0S2 genes that are known to regulate cell growth and proliferation. Treatments of human pancreatic (AsPC-1), colon (HT29), and breast adenocarcinoma cells T47D and MDA-MB-468 with the LRH-1 antagonists resulted in the receptor-mediated inhibition of cancer cell proliferation. Our data suggest that specific antagonists of LRH-1 could be used as specific molecular probes for elucidating the roles of the receptor in different types of malignancies.

Structural basis of coactivation of liver receptor homolog-1 by ß-catenin.

We report the three-dimensional structure of a ß-catenin armadillo repeat in complex with the liver receptor homolog-1 (LRH-1) ligand binding domain at 2.8 Å resolution as the first structure of ß-catenin in complex with any nuclear receptor. The surface of ß-catenin that binds LRH-1 partly overlaps defined contact sites for peptide segments of ß-catenin partners, including T-cell factor-4. The surface of LRH-1 that engages ß-catenin is comprised of helices 1, 9, and 10 and is distinct from known interaction surfaces of LRH-1, including corepressor and coactivator binding sites. Targeted mutagenesis of amino acids forming both sides of the LRH-1/ß-catenin interface reveals that they are essential for stable interactions between these proteins in solution. The LRH-1 binding site in ß-catenin is also required for association with androgen receptor, providing evidence that the observed LRH-1/ß-catenin interaction may be prototypic.

Nuclear receptor liver receptor homologue 1 (LRH-1) regulates pancreatic cancer cell growth and proliferation.

An essential regulator of gene transcription, nuclear receptor liver receptor homologue 1 (LRH-1) controls cell differentiation in the developing pancreas and maintains cholesterol homeostasis in adults. Recent genome-wide association studies linked mutations in the LRH-1 gene and its up-stream regulatory regions to development of pancreatic cancer. In this work, we show that LRH-1 transcription is activated up to 30-fold in human pancreatic cancer cells compared to normal pancreatic ductal epithelium. This activation correlates with markedly increased LRH-1 protein expression in human pancreatic ductal adenocarcinomas in vivo. Selective blocking of LRH-1 by receptor specific siRNA significantly inhibits pancreatic cancer cell proliferation in vitro. The inhibition is tracked in part to the attenuation of the receptor's transcriptional targets controlling cell growth, proliferation, and differentiation. Previously, LRH-1 was shown to contribute to formation of intestinal tumors. This study demonstrates the critical involvement of LRH-1 in development and progression of pancreatic cancer, suggesting the LRH-1 receptor as a plausible therapeutic target for treatment of pancreatic ductal adenocarcinomas.

Structure of SF-1 bound by different phospholipids: evidence for regulatory ligands.

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.

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

The Dax-1 protein is an enigmatic nuclear receptor that lacks an expected DNA binding domain, yet functions as a potent corepressor of nuclear receptors. Here we report the structure of Dax-1 bound to one of its targets, liver receptor homolog 1 (LRH-1). Unexpectedly, Dax-1 binds to LRH-1 using a new module, a repressor helix built from a family conserved sequence motif, PCFXXLP. Mutations in this repressor helix that are linked with human endocrine disorders dissociate the complex and attenuate Dax-1 function. The structure of the Dax-1:LRH-1 complex provides the molecular mechanism for the function of Dax-1 as a potent transcriptional repressor.

Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1.

Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members-mouse and human SF-1 and human LRH-1-which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.

Crystal structure of kinesin regulated by Ca(2+)-calmodulin.

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.

Structural basis for ligand-independent activation of the orphan nuclear receptor LRH-1.

The orphan nuclear receptors SF-1 and LRH-1 are constitutively active, but it remains uncertain whether their activation is hormone dependent. We report the crystal structure of the LRH-1 ligand binding domain to 2.4 A resolution and find the receptor to be a monomer that adopts an active conformation with a large but empty hydrophobic pocket. Adding bulky side chains into this pocket resulted in full or greater activity suggesting that, while LRH-1 could accommodate potential ligands, these are dispensable for basal activity. Constitutive LRH-1 activity appears to be conferred by a distinct structural element consisting of an extended helix 2 that provides an additional layer to the canonical LBD fold. Mutating the conserved arginine in helix 2 reduced LRH-1 receptor activity and coregulator recruitment, consistent with the partial loss-of-function phenotype exhibited by an analogous SF-1 human mutant. These findings illustrate an alternative structural strategy for nuclear receptor stabilization in the absence of ligand binding.

Structure of an F-actin trimer disrupted by gelsolin and implications for the mechanism of severing.

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.