Identification of natural ligands of retinoic acid receptor-related orphan receptor alpha ligand-binding domain expressed in Sf9 cells--a mass spectrometry approach

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.     

Orphan nuclear receptors adopted by crystallography

Of the large nuclear hormone receptor superfamily of proteins, orphan nuclear receptors have remained a mystery owing to their lack of identified ligands and their constitutive nature. Now, structures of several ligand-binding domains of orphan receptors have provided some surprising insights that were not anticipated from molecular studies. Therefore, most orphan nuclear receptors have now been 'adopted' and their regulation has been shown to range from true ligand-independence to highly promiscuous ligand-dependence. Former orphan receptors have been found to contain ligand-binding pockets that range in volume from vast (>1600A3) to non-existent and have been shown to generate surface AF2 motifs that range from being multifunctionally active to distinctly inactive. Insights from these new structures illustrate how powerful a structural biology approach can be when integrated with molecular and cellular physiology.     

Probing conformational changes in orphan nuclear receptor: the NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.     

Structural insights into ligand-binding pocket formation in Nurr1 by molecular dynamics simulations

Abstract

The nuclear receptor Nurr1 (NR4A2) has been identified as a potential target for the treatment of Parkinson’s disease. In contrast to most other nuclear receptors, the X-ray crystal structure of the Nurr1 ligand-binding domain (LBD) lacks any ligand-binding pocket (LBP). However, NMR spectroscopy measurements have revealed that the known Nurr1 agonist docosahexaenoic acid (DHA) binds to a region within the LBD that corresponds to the classical NR ligand-binding pocket (LBP). In order to investigate the structural dynamics of the Nurr1 LBD and to study potential LBP formation, the conformational space of the receptor was sampled using a molecular dynamics (MD) simulation. Docking of DHA into 50,000 LBD structures extracted from the simulation revealed the existence of a transient LBP that is capable to fully harbor the compound. The location of the identified pocket overlaps with the ligand-binding site suggested by NMR experiments. Structural analysis of the protein-ligand complex showed that only modest structural rearrangements within the Nurr1 LBD are required for LBP formation. These findings may support structure-based drug discovery campaigns for the development of receptor-specific agonists.     

Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains

The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.     

Farnesyl pyrophosphate is a novel transcriptional activator for a subset of nuclear hormone receptors

In silico docking of a chemical library with the ligand-binding domain of thyroid hormone nuclear receptor-beta (TRbeta) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative-activating receptors, farnesoid X receptor, liver X receptor, or several orphan nuclear receptors the ligands of which are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo, and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor ligand-binding domain structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors the ligands of which have not yet been identified. This finding also has potential interesting implications that may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells.     

Ligand binding is without effect on complex formation of the ligand binding domain of the ecdysone receptor (EcR)

The ligand-binding domain (LBD) encompassing the C-terminal parts of the D- and the complete E-domains of the ecdysteroid receptor (EcR) fused to Gal4(AD) is present in two high molecular weight complexes (600 and 150 kDa) in yeast extracts according to size exclusion chromatography (Superdex 200 HR 10/30). Hormone binding is mainly associated with 150-kDa complexes. Complex formation is not influenced by hormone, but the ligand stabilizes the complexes at elevated salt concentrations. Mutational analysis of Gal4(AD)-EcR(LBD) revealed that formation of 600-kDa, but not 150-kDa, complexes depends on dimerization mediated by the EcR(LBD). Deletion of helix 12 is without effect. Mutation of K497 in helix 4, known to be essential for comodulator binding, abolishes 600-KDa complexes, but does not interfere with the formation of 150-kDa complexes. In contrast, the DE-domains of USP fused to Gal4(DBD) elute as monomer after elimination of the dimerization capacity of the ligand-binding domains by mutation of P463 in helix 10. The data presented here reveal that the complex formation of ligand-binding domains EcR and USP ligand is different.