Nuclear Receptor Ligand-binding Domain Research Articles

Heterodimeric Complex of RAR and RXR Nuclear Receptor Ligand-Binding Domains: Purification, Crystallization, and Preliminary X-Ray Diffraction Analysis

Both the human retinoic acid receptor α (hRARα) and a constitutively active mutant (F318A) of the mouse retinoid X receptor α (mRXRαF318A) ligand-binding domains were separately overexpressed in Escherichia coli, copurified as a heterodimer in a two-step procedure, and cocrystallized with an RARα-specific antagonist by using polyethylene glycol 10,000 as precipitant. The crystals grew in the hexagonal space group P6122 displaying the unit cell parameters a = b = 116.6 Å and c = 207.8 Å. They diffracted X-ray to a limit of 2.2-Å resolution. The asymmetric unit comprises one heterodimer and the crystal contains 60% solvent. The structure was determined by molecular replacement and is currently being refined.

The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes.

Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D(3) receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARgammaLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.

Ligand Selectivity by Nuclear Hormone Receptors

Numerous crystal structures of nuclear receptor ligand binding domains (LBDs) are known. The retinoic acid (RAR) and estrogen (ER) receptors are the two members for which a large set of agonists and antagonist complexes are available. Their analysis reveals key features of the RAR and ER ligand binding pocket (LBP) responsible for ligand selectivity. The RAR LBD exhibits a rigid architecture to which the ligand has to adapt, whereas the ER LBD can accomodate numerous ligands of variable shapes.

Temperature dependence of estrogen binding: importance of a subzone in the ligand binding domain of a novel piscine estrogen receptor

The full length estrogen receptor from Oreochromis aureus (OaER) was cloned and expressed in vitro and in vivo as a functional transcription factor. Amino acid residues involved in the thermal stability of the receptor are located at/near subzones β1 and β3, which are highly conserved in other non-piscine species but not in OaER. Hormone binding studies, however, indicate that OaER is thermally stable but exhibited a ∼3-fold reduced affinity for estrogen at elevated temperatures. Transfection of OaER into various cell lines cultured at different temperatures displayed a significant estrogen dose-response shift compared with that of chicken ER (cER). At 37°C, OaER requires ∼80-fold more estrogen to achieve half-maximal stimulation of CAT. Lowering of the incubation temperature from 37°C to 25°C or 20°C resulted in a 4-fold increase in its affinity for estrogen. The thermally deficient transactivation of OaER at temperatures above 25°C was fully prevented by high levels of estrogen. Thus, compared to cER, the OaER exhibits reduced affinity for estrogen at elevated temperature as reflected in its deficient transactivation capability. Amino acid replacements of OaER β3 subzones with corresponding amino acids from cER could partially rescue this temperature sensitivity. The three-dimensional structure of the OaER ligand binding domain (LBD) was modelled based on conformational similarity and sequence homology with human RXRα apo, RARγ holo and ERα LBDs. Unliganded and 17β-estradiol-liganded OaER LBD retained the overall folding pattern of the nuclear receptor LBDs. The residues at/near the subzone β3 of the LBD constitute the central core of OaER structure. Thus, amino acid alteration at this region potentially alters the structure and consequently its temperature-dependent ligand binding properties.

Nuclear-receptor ligands and ligand-binding domains.

The determination of several structures of nuclear receptor ligand binding domains (LBD) has led to new insights into the mechanism of action of this very important class of receptors. This review describes and compares the different LBD structures and their relationship to the function of the nuclear receptors. The role of the ligand in the LBD structures and the implications of ligand structure on receptor activity are also discussed. Structural information regarding interactions between the LBD and coactivator proteins and the potential role of these interactions in ligand agonism and antagonism is reviewed. Different pathways for nuclear receptor signaling and the use of new ligands to investigate these pathways are also described.

Evolutionary constraint networks in ligand-binding domains: an information-theoretic approach.

Ligand-binding sites in homologous protein domains can diverge greatly during evolution. This poses a particularly interesting problem in those cases where the ligand-binding site is situated in, or close to, the domain core, or where ligand-docking induces dramatic conformational changes. These features are present in many receptors and enzymes; the hormone-binding domain of the nuclear receptors for steroids and retinoids, for example, exhibits both characteristics. It is therefore of great interest to determine how binding sites for diverse ligands evolve in core regions of structurally dynamic domains. Are evolutionary changes locally restricted to the ligand-binding site, or are they distributed throughout the domain? We describe here an information-theoretic approach for the study of covariation between ligand-contacting residues and compensatory mutations that preserve the structural integrity and the conformational dynamics of ligand-binding domains. We apply this method to the analysis of the nuclear receptor ligand-binding domain and show that the ligand-contacting residues in the hormone-binding pocket are evolutionarily linked to an extensive network of covarying positions.

Crystal Structure of the Ligand Binding Domain of the Human Nuclear Receptor PPARγ

The peroxisome proliferator-activated receptors (PPAR) are members of the nuclear receptor supergene family and are considered as key sensors of both lipid and glucose homeostasis. The role of the PPARgamma isoform in glucose metabolism is illustrated by the fact that anti-diabetic thiazolidinediones have been shown to be bona fide PPARgamma ligands. Here we report the crystal structure of apo-PPARgamma ligand binding domain (LBD) determined to 2.9-A resolution. Although the structure of apo-PPARgamma-LBD retains the overall fold described previously for other nuclear receptor LBDs, three distinct structural differences are evident. 1) The core AF-2 activation domain of apo-PPARgamma LBD is folded back toward the predicted ligand binding pocket similar to that observed in the holo-forms of other nuclear receptors. 2) The proposed ligand binding pocket of apo-PPARgamma-LBD is larger and more accessible to the surface in contrast to other LBDs. 3) The region of the LBD called the omega-loop is extended in PPARgamma and contains additional structural elements. Taken together, the apo-PPARgamma-LBD structure is in several aspects different from previously described LBDs. Given the central role of PPARgamma as a mediator in glucose regulation, the structure should be an important tool in the development of improved anti-diabetic agents.

Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer.

Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcriptional activation. Here we used fluorescence resonance energy transfer (FRET) as an approach for detecting and quantitating such interactions. Using the ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARgamma) as a model, known agonists (thiazolidinediones and delta12, 14-PGJ2) induced a specific interaction resulting in FRET between the fluorescently labeled LBD and fluorescently labeled coactivators [CREB-binding protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific energy transfer was dose dependent; individual ligands displayed distinct potency and maximal FRET profiles that were identical when results obtained using CBP vs. SRC-1 were compared. In addition, half-maximally effective agonist concentrations (EC59s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPARgamma (E471A) that abrogated ligand-stimulated transcription in transfected cells also failed to induce ligand-mediated FRET between PPARgamma LBD and CBP or SRC-1. Using estrogen receptor (ERalpha) as an alternative system, known agonists induced an interaction between ERalpha LBD and SRC-1, whereas ER antagonists disrupted agonist-induced interaction of ERalpha with SRC-1. In the presence of saturating agonist concentrations, unlabeled CBP or SRC-1 was used to compete with fluorescently labeled coactivators with saturation kinetics. Relative affinities for the individual receptor-coactivator pairs were determined as follows: PPARgamma-CBP = ERalpha-SRC-1 > PPARgamma-SRC-1 >> ERalpha-CBP. 1) FRET-based coactivator association is a novel approach for characterizing nuclear receptor agonists or antagonists; individual ligands display potencies that are predictive of in vivo effects and distinct profiles of maximal activity that are suggestive of alternative receptor conformations. 2) PPARgamma interacts with both CBP and SRC-1; transcriptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactivators; thus, PPARgamma has a greater apparent affinity for CBP than for SRC-1, whereas ERalpha interacts preferentially with SRC-1 but very weakly with CBP.

Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor-γ

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a ligand-dependent transcription factor that is important in adipocyte differentiation and glucose homeostasis and which depends on interactions with co-activators, including steroid receptor co-activating factor-1 (SRC-1). Here we present the X-ray crystal structure of the human apo-PPAR-gamma ligand-binding domain (LBD), at 2.2 A resolution; this structure reveals a large binding pocket, which may explain the diversity of ligands for PPAR-gamma. We also describe the ternary complex containing the PPAR-gamma LBD, the antidiabetic ligand rosiglitazone (BRL49653), and 88 amino acids of human SRC-1 at 2.3 A resolution. Glutamate and lysine residues that are highly conserved in LBDs of nuclear receptors form a 'charge clamp' that contacts backbone atoms of the LXXLL helices of SRC-1. These results, together with the observation that two consecutive LXXLL motifs of SRC-1 make identical contacts with both subunits of a PPAR-gamma homodimer, suggest a general mechanism for the assembly of nuclear receptors with co-activators.

Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors.

The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.

The nuclear receptor ligand-binding domain: structure and function

In the past few years our understanding of nuclear receptor action has dramatically improved as a result of the elucidation of the crystal structures of the empty (apo) ligand-binding domains of the nuclear receptor and of complexes formed by the nuclear receptor's ligand-binding domain bound to agonists and antagonists. Furthermore, the concomitant identification and functional analysis of co-regulators (transcriptional intermediary factors [TIFs], comprising co-activators and co-repressors) previously predicted from squelching studies, have deepened this understanding. Recent data have provided the structural basis for the specific recognition of ligands and the molecular mechanisms of agonism and antagonism, enabling us to gain a comprehensive view of the early steps of nuclear receptor action.

Steroid receptors at the nexus of transcriptional regulation.

During the past few years, our understanding of nuclear receptor action has dramatically improved as a result of the identification and functional analysis of co-regulators such as factors involved in chromatin remodeling, transcription intermediary factors (co-repressors and co-activators), and direct interactions with the basal transcriptional machinery. Furthermore, the elucidation of the crystal structures of the empty ligand-binding domains of the nuclear receptor and of complexes formed by the nuclear receptor's ligand-binding domain bound to agonists and antagonists has contributed significantly to our understanding of the early events of nuclear receptor action. However, the picture of hormone- and hormone receptor-mediated mechanisms of gene regulation remain incomplete and extremely complicated when one also considers the "nontraditional" interactions of hormone-activated nuclear receptors, for example, interactions between the activated steroid receptors and components of the chromatin/nuclear matrix; and finally the nongenomic effects that steroid hormones can exhibit with other signaling pathways. In this prospectus on steroid receptors, we discuss the implications of various steroid hormone and nuclear receptor interactions and potential future directions of investigation. J. Cell. Biochem. Suppls. 30/31:185-193, 1998. © 1999 Wiley-Liss, Inc.

A Conditional Version of the Ets Transcription Factor Erm by Fusion to the Ligand Binding Domain of the Oestrogen Receptor

The fusion of a wide range of proteins to the ligand-binding domain of nuclear receptors has been shown to impart ligand-dependent inducible activity of the resulting chimera. Transcriptional regulators of the ETS family are involved in both normal and oncogenic processes. In order to address the role of Erm, a “PEA3subgroup” member of this family, we generated a chimera between Erm and the widely used ligand-binding domain of the oestrogen receptor (ER). The chimera, ErmER, consists of Erm protein fused at its C-terminal end to the ER domain. We show that ErmER displays a ligand-dependent transcriptional activity onetsresponsive elements. The efficiency of ErmER mediated transactivation is modulated by the hormone concentration while its weak leakiness is reduced by using the steroidal anti-oestrogen EM-139. Our results define ErmER as the first conditional version of an Ets transcription factor, providing a useful tool to decipher Erm biological role and to identify potential Erm target genes.

Regulation of nuclear receptors by agonists and antagonists

The nuclear receptor superfamily presents an exceptional spectrum of targets for pharmacologic developments. Different members of the superfamily mediate the biological activities of the steroid and thyroid hormones and the vitamins A and D, among others. The pharmacologic potential of the nuclear receptors rests in part on the ability of synthetic derivatives of these hormones and vitamins to completely block, or partially imitate, the activities of the authentic ligand. The activities of these synthetic ligands pose two questions. How do some of them block the action of their cognate receptor? How do others elicit a subset of cognate receptor responses? The recent determination of crystal structures of three nuclear receptor ligand-binding domains presents structural answers to these questions. Ligand-binding domains bind ligand by burying them into the hydrophobic core of the domain. From this position, ligands can remodel the ligand-binding domain conformation. Different ligand-binding domain conformations that reflect differences in the bound ligand can explain the different biological activities elicited. This proposition is related to recent progress in the biology of ligand regulation of nuclear receptor ligand-binding domain activities.

Mutation of isoleucine 747 by a threonine alters the ligand responsiveness of the human glucocorticoid receptor.

Mutation of isoleucine 747 to threonine in the C-terminal part of the ligand-binding domain (LBD) of the human glucocorticoid receptor (GR) alters the ligand specificity for transactivation. Natural glucocorticoids such as cortisol or corticosterone were completely inactive with the mutant 1747T, whereas synthetic steroids like dexamethasone efficiently stimulated GR 1747T-mediated transactivation. However, the corresponding ligand dose-response curve for dexamethasone-induced transactivation was shifted to higher concentrations when compared with that obtained with the wild type GR. Neither this shift nor the inability of cortisol to activate the 1747T mutant was due to an altered in vitro ligand-binding affinity. In the canonical three-dimensional structure of nuclear receptor LBDs, isoleucine 747 is in the direct vicinity of residues that contribute to the ligand-binding pocket. Moreover, it is located in the C-terminal LBD region, which harbors the conserved core of the activation function AF-2 and undergoes a ligand-induce transconformation, required to generate the surface interacting with putative transcriptional intermediary factors/coactivators of AF-2. The phenotype of 1747T mutant is discussed in view of the possible consequences of the mutation on the various events which, according to the model, lead to a transcriptionally competent AF-2.

Nuclear receptors spring into action

The ligand-binding domain of nuclear receptors appears to contain a common fold that generates a conserved ligand-binding pocket. Their transcriptional activity is induced by ligand through realignment of a Gterminal helix to form a novel interacting surface.

A canonical structure for the ligand-binding domain of nuclear receptors

The ability of nuclear receptors (NRs) to activate transcription of target genes requires the binding of cognate ligands to their ligand-binding domains (LBDs). Information provided by the three-dimensional structures of the unliganded RXR alpha and the liganded RAR gamma LBDs has been incorporated into a general alignment of the LBDs of all NRs. A twenty amino-acid region constitutes a NR-specific signature and contains most of the conserved residues that stabilize the core of the canonical fold of NR LBDs. A common ligand-binding pocket, involving predominantly hydrophobic residues, is inferred by homology modelling of the human RXR alpha and glucocorticoid receptor ligand-binding sites according to the RAR gamma holo-LBD structure. Mutant studies support these models, as well as a general mechanism for ligand-induced activation deduced from the comparison of the transcriptionally active RAR gamma holo- and inactive RXR alpha apo-LBD structures.

Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-α

The crystal structure of the human retinoid-X receptor RXR-alpha ligand-binding domain reveals a previously undiscovered fold of an antiparallel alpha-helical sandwich, packed as dimeric units. Two helices and one loop form the homodimerization surface, and hydrophobic heptad repeats participate in stabilizing the fold. The existence of a ligand-binding pocket is proposed that would allow 9-cis retinoic acid to interact with different functional modules, including the AF-2 activating domain. Several lines of evidence indicate that the overall structure is a prototype fold of ligand-binding domains of nuclear receptors.