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Available online at www.sciencedirect.com

Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54

Structural determination of estrogen-related receptor �in the presence of phenol derivative compounds

Marta C. Abad ∗, Hossein Askari, John O’Neill, Alexandra L. Klinger, Cynthia Milligan,Frank Lewandowski, Barry Springer, John Spurlino, Dionisios Rentzeperis

Johnson & Johnson Pharmaceuticals Research and Development, 665 Stockton Drive, Exton, PA 19341, USA

Received 21 March 2007; accepted 27 June 2007

bstract

We screened the ligand-binding domain of estrogen-related receptor (ERR) � in ThermoFluor®, in an effort to develop chemical toolsnd decipher the biology of this orphan nuclear receptor. Several ligands were found to stabilize thermodynamically the protein. Amongsthe ligands were bisphenol A (BPA) and 4-chloro-3-methyl phenol (ClCH3Ph). These ligands were further characterized and found to beompetitive for 4-hydroxytamoxifen (4OHT) binding, a known reported antagonist ligand for ERR�, but functionally they did not enhancer disrupt affinity of the receptor for co-activator peptides. The preservation of the constitutive active conformation of the receptor in theresence of these two ligands was confirmed upon the determination of the co-crystal structures. The structures of BPA and ClCH3Ph wereetermined to a resolution of 2.1 and 2.3 A, respectively, and the antagonist 4OHT was refined to 2.5 A resolution. In the presence of BPAnd ClCH3Ph the receptor maintained the transcriptional active conformation as reported previously for the apo-protein in the presence of a

o-activator peptide fragment. In addition the ERR�-BPA structure identifies an interaction between the phenolic-OH and the side chain of346. The preservation of the constitutive active conformation of the receptor in the presence of the small phenol compounds suggest that

he biological activity of the receptor might be regulated by a natural occurring ligand.ublished by Elsevier Ltd.

henol; 4

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eywords: Estrogen-related receptor �; Bisphenol A; 4-Chloro-3-methyl p

. Introduction

Nuclear hormone receptors (NHRs) play key roles inevelopment, homeostasis, and disease [1–3]. Targeted geneeletion of these receptors in mice has proven their asso-iation with different diseases including atherosclerosis,ancer, diabetes, and lipid disorders [4–6]. These find-

ngs have opened new strategies for treatment of theseiseases, and implicate orphan receptors as important tar-ets for drug discovery. Nuclear receptors (NRs) act as

Abbreviations: 4OHT, 4-hydroxy-tamoxifen; BPA, bisphenol A;lCH3Ph, 4-chloro-3-methyl phenol; DES, di-ethylstilbestrol; ER, estro-en receptor; ERR, estrogen-related receptor; H12, helix 12; �Hb, bindingnthalpy; HTS, high throughput screening; LBD, ligand-binding domain;BP, ligand-binding pocket; ND, not determined; NHR, nuclear hormone

eceptors; NR, nuclear receptor; TR-FRET, time-resolved fluorescence res-nance energy transfer; SRC, steroid receptor co-activator.∗ Corresponding author. Tel.: +1 610 458 5264; fax: +1 610 458 8249.

E-mail address: mabad@prdus.jnj.com (M.C. Abad).

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960-0760/$ – see front matter. Published by Elsevier Ltd.oi:10.1016/j.jsbmb.2007.06.006

-Hydroxy-tamoxifen

igand-inducible transcription factors that are regulatedy binding to small lipophilic molecules such as steroidnd thyroid hormones or the active forms of vitamin Aretinoids) and vitamin D [7–10]. These molecules playn important role in the embryonic development, growth,ifferentiation, metabolism, reproduction, homeostasis andorphogenesis of higher organisms and humans. Severalembers of the nuclear receptor family for which ligands

ave not been identified are classified as orphan receptors11,12].

The estrogen-related receptors (ERRs) were the firstrphan NRs to be discovered and to date three membersave been identified (ERR�, ERR� and ERR�). The ERRubfamily is closely related to the estrogen receptors ER�nd ER�. ERR� and ERR� were first isolated by a low

tringency hybridization screen [13] followed later with theiscovery of ERR� [14]. Though sharing structural homol-gy with the estrogen receptors, these receptors do not bindstrogens. Unlike classical estrogen receptors that are ligand-

emistry

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M.C. Abad et al. / Journal of Steroid Bioch

ctivated NRs, the ERR’s show varying levels of constitutivectivity that appears to be tissue selective [6,15]. The ERRsnd ERs share sequence similarity with the highest homol-gy observed in their DNA binding domains. They interactith classical DNA estrogen response elements and half sites

16,17]. Recent biochemical evidence has shown that theRRs and ERs share co-regulatory proteins and can func-

ionally interfere with estrogen responsive genes in the breastnd bone including pS2, lactoferin, aromatase and osteopon-in [14,15,17–19]. In addition ERR� is positively correlatedith ER� positive breast cancers [20] and is associated with aositive prognostic outcome for anti-estrogen therapies. Theater might be due that 4OHT is a potent ligand for ERR� [21].

ERR� is highly expressed in metabolic active tissues dur-ng fetal development such as skeletal muscle, adipose andeart in a similar manner to ERR� [22]. In the adult high-st expression levels are observed in the heart, brain, kidneynd pancreas. GST-pulldown experiments have shown thatRR� physically interacts with PGC-1 and RIP-140, both ofhich are known nuclear receptor co-regulators [23]. Its basal

ranscriptional activity is strongly stimulated in the presencef the PGC-1 family of transcription factors, while RIP-140epresses its activity. PGC-1 has an important role in glu-oneogenesis, thermogenesis and mitochondrial biogenesis,hile RIP-140 is a suppressor of mitochondrial biogenesis

nd a negative regulator of energy expenditure [24]. Theact that both co-regulators and ERR� have similar tissuexpression profiles, relative protein levels can impact biolog-cal activity of the receptor implicating ERR� as a regulatorf energy homeostasis [25].

One of the rate limiting steps in defining biological func-ion for orphan NRs is the discovery of interacting ligandshat would pharmacologically modulate its biological activ-ty. New screening technologies have been developed for theiscovery of ligands for orphan NHRs, and have assisted inhe identification of ligands that can be used as tools for eluci-ating the biology of these receptors [26,27]. This approachs referred to as “reverse endocrinology” [28]. Although

high-risk endeavor, the ERR NRs are highly druggables several reports have attested to in recent years. Phar-acological modulation of ERR� by the small molecule

ntagonist XCT790 has elucidated the role of ERR� inhe regulation of oxidative phosphorylation genes [29,30].iochemical and X-ray crystallography studies of 4OHTnd di-ethylstilbestrol (DES) showed that ERR� and ERR�re antagonists and the determined co-crystal structures ofRR� provided the molecular basis of the observed antago-ism [21,31]. Phenolic acyl hydrazones have been describeds ERR� agonists although no details were given on theolecular basis for the observed agonist response [32]. We

ave initiated an effort in identifying ligands that stabi-ize the ligand-binding domain for all three members of

he ERR family using ThermoFluor® as a high through-ut screening (HTS) platform [33]. ThermoFluor® exploitshe well-characterized phenomenon of ligand-induced stabi-ization of macromolecules [34]. The technology provides

csEfi

& Molecular Biology 108 (2008) 44–54 45

ompetitive advantages over existing technologies since itoes not require a functional response and can detect lowffinity ligands [35,36].

In this report we describe the identification of compoundshat associate and stabilize the ligand-binding domain ofRR�. Affinities for these ligands were measured by isother-al methods and their functional response was determined

y a co-activator recruitment assay. Two of the identified phe-ol containing ligands, BPA and ClCH3Ph, associates withotencies of 70 and 380 nM, respectively, and competes forOHT binding, a reported antagonist of ERR�. The struc-ures for these two compounds were determined to resolutionsf 2.1 and 2.3 A, respectively. Superimposition of the struc-ures with the reported constitutively active apo-form of theeceptor showed no changes in the overall conformation ofhe receptor consistent with the ligands being functionallyilent.

. Materials and methods

.1. Cloning, expression and purification

Human ERR� ligand-binding domain (LBD) (229–458)as subcloned in a pET28a vector (Novagen). The vectoras transformed in E. coli BL21(DE3) and cultured in Min-

mal media (M9) at 37 ◦C to an OD595 0.6–0.8, followed bynduction at 15 ◦C with 0.4 mM IPTG for 20 h. The next day,he culture was harvested and lysed in buffer A (buffer A:0 mM Tris–HCl, pH 7.5, 0.4 M NaCl and 5 mM imidazole)sing an Emulsiflex-C5 French press (Avestin). Lysates werepun at 40,000 × g for 45 min to remove insoluble material.he clarified lysate was applied on a Ni-NTA column andluted with a linear gradient of imidazole. The protein wasurther purified by applying the eluent on a Superdex 200 HR0/30 column (Amersham Pharmacia Biotech) equilibratedith buffer B (buffer B: 20 mM Tris–HCl pH 7.5, 0.2 M NaCl,mM EDTA and 1 mM DTT). The aggregation state of therotein was confirmed by dynamic light scattering. Fractionsere pulled, concentrated to 4 mg/mL and frozen in liquiditrogen and stored at −80 ◦C.

.2. ThermoFluor® screen and determination of bindingonstants

ThermoFluor® is an HTS assay that measures proteinnfolding based on fluorescence detection of the denaturedorm of the protein. Details on this HTS screening platformnd the mathematical models used to derive melting tem-eratures and equilibrium binding constants have appearedlsewhere [33]. Briefly assay plates were prepared by dis-ensing 2 �L of a protein–dye solution and 2 �L of the test

ompound in a 384-well plate. The conditions used in thecreen were 25 mM HEPES pH 8.0, 100 mM NaCl, 1 mMDTA, 0.1 mg/mL ERR�, 25 �M ANS, 2% DMSO and thenal concentration of the test compound was 0–200 �M.

4 emistry

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6 M.C. Abad et al. / Journal of Steroid Bioch

inally, 1 �L of mineral oil was dispensed on top to preventvaporation during the HTS.

For the ERR� screen, data were collected from 30 to 80 ◦Ct 1 ◦C intervals and the protein melted under the screeningonditions with a characteristic melting temperature, Tm, of9.0 ◦C. Hits were identified from the screen by measuringn increase in the melting temperature of the protein. For theinding affinities it was assumed that ERR� is a dimer andhat a single ligand can interact with an equal affinity per

onomer subunit.In the thermodynamic treatment of the data the follow-

ng assumptions were made: (i) the small ligand interactsnly with the folded state of the protein; (ii) the reactions areeversible; (iii) the unfolding protein reaction is a two-staterocess; and (iv) ideal dilute solutions are being used (spe-ific activity for protein and ligands is equal to 1). All fittingnd numerical integrations were done using the commercialrogram MicroMath® Scientist® version 2.01 (MicroMathcientific Software, Salt Lake City, Utah).

.3. TR-FRET assay

Time-resolved fluorescence resonance energy transferTR-FRET) experiments were performed to examine theunctional response of ERR� ligands. The components of thisomogeneous secondary assay included: the 6His-tagged-RR� LBD, a GST-labeled-hSRC2 co-activator polypeptidend a fluorescent donor/acceptor pair from CIS bio interna-ional htrf/bioassays (Bedford, MA) using both an �-GSTuropium Cryptate (Eu) label and an �6His-XL665 (allo-hycocyanin) fluorophore.

For TR-FRET measurements, the reaction was buffered in5 mM Tris pH 8, 2.5 mM Hepes, 20 mM KCl, 1 mM DTT,nd 0.05 mg/mL BSA. The final concentrations of reagentsere 6 nM of ERR� LBD, 6 nM GST-Src peptide, 30 nMu cryptate, and 7.5 nM XL665. Reactions were allowed to

each equilibrium at 25 ◦C for 18 h before collecting data onhe Analyst from LJL Biosystems (Molecular Devices Sun-yvale, CA). As a time-resolved method, the samples werexcited at 340 nM and emission was collected for 1 ms at both15 and 665 nm with delays of 400 and 75 �s, respectively.ose–response curves were fitted using a hyperbolic equa-

ion and the data reported is the average of three independentxperiments.

.4. Isothermal titration calorimetry

Binding constants for BPA and ClCH3Ph were measuredy using isothermal titration calorimetry. The heat of inter-ction of the ligands with ERR� molecules was measuredy using the Omega titration calorimeter from Microcal Inc.n a typical titration, 1.4 mL of a 20 �M protein solution

s titrated with a ligand solution of ∼0.4 mM concentrationsing a 100 �L syringe by ∼20 injections of 5–6 �L each.his syringe is also used to mix the reactants completely

n the sample cell by rotating it at 400 rpm. For each injec-

w[sw

& Molecular Biology 108 (2008) 44–54

ion, the area under the resulting peak is proportional to theeat of interaction, Q. Once this heat is corrected for theitrant dilution and normalized by the titrant solution con-entration it becomes equal to the binding enthalpy, �Hb,nder nonsaturating conditions. The precision of the heat ofach injection is about 0.5 �cal. One method of determin-ng �Hb is to average several intermediate peaks from theifferent sites. In addition to the binding enthalpies, one canbtain binding affinities, and overall stoichiometries of theomplexes. The experimental calorimetric binding isotherms the dependence of the total heat, Qr (or (dQr/dX) T) onhe total concentration of ligand added, Xr. The above threearameters for each type of site are determined iterativelysing the Marquardt algorithms as described previously. Thenitial fitting procedure lets all three parameters float or fixesither the enthalpy, determined independently by averaginghe heats of the intermediate peaks of a given site, or n param-ters or both until the lowest standard deviation of the fit isbtained; all approaches result in similar values.

.5. Crystallization, data collection and structureetermination

Purified human ERR�-LBD was buffer exchanged in0 mM Tris pH 7.5, 0.2 M NaCl, 1 mM EDTA, 1 mM DTTnd 5% glycerol, complexed with the compound of interestn a 1:2 molar ratio and concentrated to ∼11 mg/mL. Therotein was screened for crystallization using the hangingrop vapor diffusion method. The reservoir contained 650 �Lf the precipitant solution and the 2 �L hanging drop con-isted of a 1:1 protein to precipitant solution ratio. Crystalsormed at 277 K from a solution containing 22.6% polyethy-ene glycol 4000, 0.1 M Tris, pH 8.5 and 0.2 M sodiumcetate. Crystals appear and were suitable for data collectionhe next day (0.2 mm × 0.2 mm × 0.1 mm). The crystals wereransferred to a cryoprotectant solution containing 22.6%olyethylene glycol 4000, 0.1 M Tris, pH 8.5, 0.2 M sodiumcetate and 20% glycerol. The crystals were then mountednd quickly frozen by immersion in liquid nitrogen. X-rayiffraction data to a resolution of 2.1 A for BPA, 2.3 A forlCH3Ph and 2.5 A for 4OHT were collected on a BrukerXS Proteum 6000 detector. Diffraction data was indexed,

ntegrated and scaled using the Proteum Processing Programuite from Bruker AXS. Under these conditions, the crystalselong to the P41212 space group, with unit cell parame-ers listed in Table 1 for each structure. Assuming one ERR�

olecule (28.5 kDa) the crystal volume per protein mass is.5, which corresponds to approximately 49.7% solvent con-ent in the crystal. This value is within the range observed forrotein crystals.

The structure was determined by molecular replacementsing one molecule from the homo dimer structure of ERR�

ith the SRC-1 peptide (PDB id 1KV6) as the search model

37]. Multiple rounds of structure refinement, using theimulated annealing method, followed by the addition ofater molecules and resolution extension resulted in the final

M.C. Abad et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54 47

Table 1Crystal and refinement parameters

Parameter BPA ClCH3Ph 4OHT

Unit cell (A) a = b = 64.07 c = 136.48 a = b = 64.28 c = 137.18 a = b = 64.00 c = 137.95Resolution (A) 2.1 2.3 2.5Completeness (%) 98.8 (98.2) 98.9 (98.1) 92.2 (77.9)Rmerge (%)a 4.8 (14.0) 11.8 (29.2) 9.0 (30.1)〈I〉/〈σI〉 11.7 (4.3) 5.9 (1.5) 5.8 (1.5)Rfactor (%)b 21.0 20.51 21.3Rfree (%)c 26.0 25.64 26.3Bfactor,avg 18.0 10.50 21.3Rmsd bonds (A)d 0.005773 0.005881 0.007911Rmsd angles (◦) 1.06264 1.02253 1.15761

Values in parenthesis refer to the highest resolution shell.a Rmerge =

∑hkl

∑I (|II − 〈I〉|/<I>), where II is an individual intensity measurement and 〈I〉 is the average intensity for this reflection, with summation over

all data.

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efinement parameters listed in Table 1. The atomic coor-inates and structure factors (codes 2P7G for BPA, 2P7Aor ClCH3Ph and 2P7Z for 4OHT) have been deposited inhe Protein Data Bank Research Collaboratory for Struc-ural Bioinformatics, Rutgers University, New Brunswick,J (http://www.rcsb.org/).

.6. Cell-based assays

A cell-based reporter assay was used to determine theunctional response of the ERR� hits. Transfections were per-ormed in HEK293E cells that were maintained in DMEMupplemented in glutamine and 10% FBS. Co-transfectionsf 4 �g of a luciferase reporter plasmid and 4 �g of eachBIND-Gal4-ERR� and pACT-SRC2 plasmids per T-75ask were done using Lipofectamine2000 as per manufac-

urers instructions. Twenty-four hours post-transfection, theells were seeded in 96-well plates at density of 50,000 cellser well in assay media (DMEM phenol free, 5% charcoaltripped FBS). The cells were allowed to adhere to the bottomf the wells approximately 5 h post-seeding and the com-ounds were dosed and the final concentration of DMSOas kept below 0.3%. After 24 h of compound treatment,

ells were lysed and treated with the Promega Dual-Gloystem. Firefly Luciferase activity was read using a lumines-ence plate reader, and data were normalized against Renillauciferase activity. Data were fitted using subroutines avail-ble from GraphPad.

. Results

.1. Identification of ligands that stabilize ERRγ andetermination of binding affinities

ThermoFluor® is a miniaturized thermal shift assay thatonitors the fraction of unfolded protein as a function of tem-

erature. Ligand-binding energy adds directly to the ground

tTat

tate energy of a macromolecule and results in increasedhermal stability. The degree of stabilization is proportionalo the binding affinity of the ligand for the macromolecule.

e expressed and characterized biochemically and biophys-cally the ligand-binding domain of the receptor as partf our protein characterization prior to screening in thehermoFluor® platform. The protein under our experimen-

al conditions (concentration 4 �M and higher) behaves asdimer as judged by size exclusion chromatography and

ynamic light scattering with apparent molecular weights of1 and 52 kDa, respectively. Further biophysical character-zation using thermal denaturation methods by monitoringoss of �-helicity by CD spectroscopy, shows that the pro-ein melts in a cooperative fashion over a narrow temperatureange, and a positive thermal stabilization is observed in theresence of the reported antagonist 4OHT (data not shown).he protein solution conditions were further optimized forThermoFluor® screen. We measured the effect of ligands

n the thermal stability of ERR� by measuring the frac-ion of unfolded protein as a function of temperature usinghe environmentally sensitive dye ANS as a reporter that isncorporated in the screening buffer. Approximately 3000ompounds were screened against this receptor. The col-ection of compounds screened contained a generic drugollection, bioactive molecules (fatty acids and steroids)nd known estrogenic compounds. Several compounds wereound to produce a positive stabilization of the receptor up to◦C (Fig. 1) and a subset of them is reported in Table 2.

The affinity of the respective ligands against this nucleareceptor panel was calculated as described previously33–36] and additional details on the thermodynamic parame-ers are provided in the footnote of Table 3. These calculationsake into account that the melting of the NHR’s is a two-staterocess and that we are measuring the denaturation of a dimer

o an unfolded protein and that the reaction is reversible.he determined binding constants calculated in such wayre estimates and reflect the induced ligand stabilization ofhe receptors. These values are provided so that meaningful

48 M.C. Abad et al. / Journal of Steroid Biochemistry

Fig. 1. Representative thermal denaturation curves of ERR� inThermoFluor®. ERR� (4 �M protein in 25 mM HEPES pH 8.0, 100 mMNaCl, 1 mM EDTA, 25 �M ANS and 2% DMSO) melted with at a charac-teristic temperature of 49.6 ◦C in the absence of ligand ( ). In the presenceof ClCH3Ph (�), BPA (�) or 4OHT (�) at 100 �M the protein stabilityincreased from 4.0 to 5.7 ◦C.

Table 2Thermal stabilization of ERR’s and ER’s against the Functional ProbeLibrarya

Ligand class ERR� ERR� ERR� ER� ER�

Phenol/polyphenolBPA 0.0 −0.1 5.5 4.4 7.4ClCH3Ph 0.1 0.2 3.9 0.4 2.44-Bromocresol 0.1 0.5 4.3 ND ND4-Chloro-3-ethyl-phenol 0.2 0.4 4.1 ND ND

Steroid17-�-Estradiol 0.1 −0.1 0.6 14.8 17.517-�-Estradiol −0.1 0.0 1.3 10.4 12.82-Methoxy-estradiol 0.1 −0.1 0.0 3.5 2.9Estrone 0.0 −0.2 0.8 7.7 11.317-�-Ethynyl-estradiol 0.3 −0.4 1.6 15.5 15.34-Estrene-3-�-17-�-diol 0.0 −0.1 −0.1 4.9 7.7

Small synthetic ligandsDES −0.1 0.5 2.7 12.0 18.9Tamoxifene −0.1 −0.1 2.7 8.5 9.84OHT −0.1 2.15 5.7 17.1 18.2Clomiphene −0.1 −0.1 0.7 8.4 9.7Raloxifene 0.1 0.0 0.0 16.7 11.9DPN −0.2 0.2 0.3 5.5 11.7PPT −2.3 −3.5 −1.8 11.7 9.5Genistein 0.0 0.1 0.1 7.4 13.9Daidzein 0.0 0.3 −0.1 4.1 8.9ICI-182780 0.0 0.0 0.0 13.9 16.7Resveratrol 0.0 0.2 0.4 ND ND

a ThermoFluor® melts were performed in 25 mM HEPES pH 8.0, 100 mMNaCl, 1 mM EDTA, 0.1 mg/mL protein, 25 �M ANS, 2% DMSO and thefinal concentration of test compound was 100 �M. Melting temperatures,Tm, under these experimental conditions for ERR�, ERR�, ERR�, ER� andER�, were, 54.4, 47.5, 49.6, 39.1 and 41.1 ◦C respectively. �Tm values arethe change of melting temperature for the free protein in the presence ofexcess test compound.

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& Molecular Biology 108 (2008) 44–54

omparisons can be made among observed �Tm changes andinding constants at some reference state, since the reportedffinities are dependent on the observed induced stabilizationnd the ground state of the ligand-free receptor.

Several classes of compounds were found to stabilizeRR�. The majority of the compounds interacted weaklyith micromolar affinities (Table 2), but for some, sub-icromolar estimates were obtained; including BPA, an

ndocrine disruptor [38], ClCH3Ph, a potent activator of Ca2+

elease from the sarcoplasmic reticulum of skeletal muscle39], and the previously reported estrogen receptor antag-nist 4OHT [21]. Counter-screening against the other twoembers of the ERR subfamily (ERR� and ERR�) did not

esult in any appreciable stabilization of the receptors withhe exception of 4OHT against ERR�; and as expected allatural and synthetic estrogenic ligands stabilized ER� andR�. In addition, we found that the isoflavones daizdein andenistein did not interact with any of the ERR’s, in con-rast to a previous study where they were reported as agonistor ERR� [40]. Estradiol and derivatives stabilized ERR�arginally with 17-�-estradiol being the most potent char-

cterized by single digit micromolar bind affinity. Moras andenaud have demonstrated the weak association of estradiol

o ERR� using non-denaturing mass-spectroscopic methods37] and recently published the presence of a fortuitous co-rystallized cholate molecule in the ERR�-4OHT complex31]. The implication of the interaction of these steroid lig-nds is not known since the concentration required to interactith the receptor are supra-physiological.

.2. Hit profiling of BPA and ClCH3Ph

We employed a TR-FRET based assay to functionallyharacterize BPA and ClCH3Ph. ERR� is regarded as aonstitutive active receptor and has the ability to recruito-activators peptides in the absence of an agonist ligand.either ligand displace the co-activator peptide bound to the

eceptor, in contrast with 4OHT that displaced the peptideragment with an affinity of ∼11 nM (Fig. 2A). In the pres-nce of 4OHT, both ligands were competitive for 4OHT asonitored by the association of the peptide to receptor withC50 values of 120 and 500 nM, respectively (Fig. 2B). Bind-

ng of these ligands was also observed by isothermal titrationalorimetry, which was characterized by highly exothermicinding enthalpies (�Hb ∼ −21 kcal/mol) and affinities of 70nd 380 nM, respectively (data not shown). We were not ableo demonstrate an enhancement in the affinity of the receptoror co-activator peptide fragments or a change in transcrip-ional activity in a trans-activation cellular assays. Therefore,oth ligands are potent for ERR� and competitive for bindingt the 4OHT binding site but functionally silent.

.3. Overall X-ray structure

ERR� was co-crystallized with BPA and ClCH3Ph in anttempt to understand the molecular basis of the functional

M.C. Abad et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54 49

Table 3Estimated dissociation constants for ligands against ERR’s and ER’s receptorsa

Ligand class ERR� ERR� ERR� ER� ER�

Phenol/polyphenolBPA – – 2.97E-07 1.64E-06 1.79E-07ClCH3Ph – – 7.88E-07 3.73E-05 3.05E-064-Bromocresol – 1.72E-05 6.33E-07 ND ND4-Chloro-3-Ethyl-phenol – 1.80E-05 7.22E-07 ND ND

Steroid17-�-Estradiol – – 1.99E-05 6.87E-09 4.63E-1017-�-Estradiol – – 6.36E-06 7.62E-08 8.39E-092-Methoxy-estradiol – – – 2.66E-06 2.31E-06Estrone – – 1.33E-05 3.04E-07 2.05E-0817-�-Ethynyl-estradiol – – 4.29E-06 4.47E-09 1.86E-094-Estrene-3-�-17-�-diol – – 1.27E-06 1.57E-07

Small synthetic ligandsDES – 1.49E-05 1.73E-06 3.15E-08 1.91E-10Tamoxifene – – 1.72E-06 2.02E-07 4.89E-084OHT – 2.64E-06 2.62E-07 1.83E-09 3.00E-10Clomiphene – – 1.56E-05 2.10E-07 5.21E-08Raloxifene – – – 2.27E-09 1.46E-08DPN – – – 9.00E-07 1.65E-08PPT – – – 3.87E-08 5.83E-08Genistein – – – 3.48E-07 4.45E-09Daidzein – – – 1.87E-06 8.15E-08ICI-182780 – – – 1.13E-08 7.89E-10Resveratrol – – – ND ND

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ilent nature of these two ligands. All of our ERR� structuresrystallized in the P41212 space group with one molecule inhe asymmetric unit. A crystallographic twofold generates theunctional homo dimer observed in all previously publishedRR� structures [31,37]. The ligand-binding pocket (LBP)

s formed mostly by hydrophobic residues and surrounded byn �-helical bundle and one small beta sheet. The few polaresidues comprising the LBD include R316, E275, and N346,hich make hydrogen bond interactions with the ligands.Analysis of the determined ERR� structures shows

onserved arrangement of secondary structural elements,ompared to previously determined ERR and ER structures.n particular the H8/H9 loop is eight residues shorter inRR�, compared to ER� shifting H9 by half a turn. Oneajor difference between ERs and ERRs lies in the residue

t position 476, a Phe in ERRs and a Leu in ERs. This makeshe cavity of ERRs smaller, limiting the size of compoundshat can bind. An overlay between the ER�-estradiol struc-ure and ER-�-genistein shows that both ligands would not beble to fit in ERR�’s cavity without causing major secondarytructure rearrangement [41,42].

.4. Agonist structures

ERR� adopts an active conformation without a boundigand, a characteristic only observed in orphan nucleareceptors [37]. A close look into the active site of the ERR�-PA structure shows that the hydroxyl groups of the ligand

tpiA

rom the induced thermal stabilization values reported in Table 2. Detailsing of ERR�, ERR�, ERR�, ER� and ER� were 190, 155, 165, 112 and

re within hydrogen bonding distance of residues N3462.82 A), R316 (2.89 A), E275 (3.33 A) and weakly hydrogenonded to Y326 (3.74 A) (Fig. 3). A hydrogen bond between326 and N346 (2.78 A) holds N346 in position to interactith the second phenyl ring of BPA. The asparagine at posi-

ion 346 in ERR� is not conserved among nuclear receptorsERR� (Gly), ERR� (Tyr), ER� (Phe) and ER� (Phe)). Thissparagine makes the N346-OH interaction specific to ERR�,ompared to the other ERs and ERRs. The highly hydropho-ic pocket of ERR� provides a favorable environment for thehenyl rings and methyl groups in BPA and ClCH3Ph. Thetructure of ERR� with ClCH3Ph (Fig. 4) reveals interactionsetween the phenyl oxygen of the ligand and residues E2752.6 A) and R316 (3.43 A). This interaction is also observed inhe BPA structure. Fig. 5 illustrates the final 2Fo–Fc electronensity map of the three ligands discussed in this article.

The structures of the two functionally silent ligands,PA and ClCH3Ph, present a conserved position for thective site residues between the apo and ligand bound struc-ures (Fig. 3). An overlay of the ERR�-steroid receptoro-activator-1 (SRC) peptide (pdb id 1KV6) onto ERR�-BPAhowed no conformational change throughout the main chain.ide chain movement is observed in a few residues. Residue275 reorients to maximize hydrogen bond interactions with

he ligand. The side chain of L345 moves away from theocket upon BPA binding, opening up the cavity and mak-ng room for the second phenyl ring of BPA (Fig. 3) [37].

comparison of the residues involved in the co-activator

50 M.C. Abad et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54

Fig. 2. TR-FRET assay in the presence of ClCH3Ph, BPA or 4OHT. ForFRET measurements, the reaction was buffered in 25 mM Tris pH 8, 2.5 mMHepes, 20 mM KCl, 1 mM DTT, and 0.05 mg/mL delipidated BSA. Thefinal concentrations of reagents were 6 nM of ERR� LBD, 6 nM GST-SRC-2 peptide, 30 nM �-GST europium cryptate and 7.5 nM �6His-XL665(allophycocyanin) fluorophore. (A) Competitive displacement curves ofGST-SRC2 in the presence of increasing concentration of ligand. 4OHTantagonized the ERR�-SRC2 complex with an apparent IC50 value of 11 nM.BPA and ClCh3PH were ineffective in antagonizing the receptor co-activatorcomplex. (B) TR-FRET assay ran in the presence of 100 nM 4OHT that effec-tCdr

bpillbn

latsp

Fig. 3. Comparison between the LBPs of ERR�-BPA (yellow) and theERR�-SRC peptide (green) structures. E275 moves to make hydrogen bondinteraction with the ligand. ERRg-BPA’s ligand is depicted in gray. Ligandsand residues atoms are colored by element nitrogen (blue), oxygen (red), andchlorine (orange). Hydrogen bonds are shown by doted lines in this figureand Figs. 4 and 6a. This figure and Figs. 6 and 7 were made using RIBBONS[49].

ively inhibits the formation of the ERR�-SRC2 complex. Both BPA andlCH3Ph allowed formation of the ERR�-SRC2 complex by competitiveisplacement of 4OHT. Apparent EC50’s obtained were 120 and 500 nM,espectively.

inding in the ERR� agonist structures and the ERR�-SRCeptide structure reveals that the orientation of those residuess conserved. Moreover, the aforementioned structure over-ay between ERR�-SRC and ERR�-BPA reveals that theseigand bound structures will allow for co-activator peptideinding. This is consistent with the fact that these ligands doot enhance or disrupt co-activator binding.

A comparison between our structures and the recently pub-ished ERR� structure shows a very similar C alpha trace and

ligand-binding pocket much smaller for ERR� compared

o ERR� [43]. The cavity size in ERR� is reduced by theubstitution of two alanines (A272 and A431) in ERR� by ahenylalanine (F328) and a valine (V491), respectively. An

Fig. 4. Comparison between the structures of ERR�-BPA (yellow) andERR�-ClCH3Ph (lavender). Ligands are depicted in gray for BPA and greenfor ClCH3Ph. Figure was made using Pymol [50].

M.C. Abad et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54 51

F conto1

oB

3

pbaHo

bbstbflamw

sdssti

a4nnaLtow

G

ig. 5. Final 2Fo–Fc electron density map of the different ligands. (A) BPA.3�.

verlay of our ERR� agonist structures revealed that neitherPA nor ClCH3Ph will be able to fit in ERR�’s cavity.

.5. Antagonist binding

The X-ray structure of ERR� with 4OHT reveals the dis-lacement of H12 from the position essential for co-activatorinding resulting in an inactive protein conformation. Inter-ctions between 4OHT and ERR� are shown in Fig. 6.ydrogen bonds with E275 (2.75 A) and R316 (3.53 A), alsobserved in the agonist structures hold the ligand in position.

The antagonist mechanism of 4OHT can be explainedy comparing ligand-binding and side chain rearrangementetween this structure and the previously presented agonisttructures. The benzyl group of 4OHT pushes F435 out ofhe cavity towards Y436, making hydrophobic interactionsetween both rings and opening up the cavity to make roomor the larger ligand (Fig. 6). Once F435 reorients, it col-

ides with L454 in H12, forcing H12 to move out of itsgonist position. Moreover, the end part of the long 4OHTolecule collides with F450, also part of H12. Initially, itas thought that the long and bulky 4OHT molecule was

Hoop

ured at 1.5�. (B) ClCH3Ph contoured at 1.3� and (C) 4OHT contoured at

olely responsible for the displacement of H12, but a recentlyetermined structure of the smaller antagonist diesthylstilbe-trol, displays the same antagonistic behavior [31]. Thismaller molecule does not collide with F450. The reorien-ation of F435 suggests to be the event responsible for ERR’snhibition.

A comparison between the ERR� and 4OHT and thegonist structures presented in this study shows residues41–456, observed in the BPA and ClCH3Ph structures, areo longer observed in the 4OHT structure as it is part of theow disordered H12. Also, the side chain of L345 adapts to fitdifferent size of ligand. The ERR�-4OHT structure shows345 moving toward the empty space previously occupied by

he second phenol ring of BPA. The shifting of the side chainf L345 is also observed in the Apo ERR�-SRC structure andith the smaller ligand, ClCH3Ph.The previously published ERR�-4OHT structure by

reschik’s et al. presented an ERR�-4OHT structure with

12 packed against the LBP of a neighboring molecule unlikeur structure, which has a disordered H12 [31]. His analysisf the structure indicated that this orientation is due to crystalacking and has no physiological significance. Our structure

52 M.C. Abad et al. / Journal of Steroid Biochemistry & Molecular Biology 108 (2008) 44–54

Fig. 6. (A) Some of the ERR� residues that delineate the LBP. Hydrogenbonds between the phenyl group and E275 and R316 and between the amineand D273 hold the ligand in position. ERR�-4OHT residues shown in laven-der; ERR�-BPA residues in yellow and 4OHT ligand in green with atomscolored by element. (B) Overlay of the LBPs of ERR�-BPA over ERR�-4OHT. Residues F450 and L454 not observed in the ERR�-4OHT structureare part of H12. Diagram shows collision of ligand with F435 and F450 andof residue F435 with L454. Side chain of residue D273 reorients to makecontact with 4OHT’s amine.

Fig. 7. Overlay of the LBPs of ER�-4OHT over ERR�-4OHT. The F435Lmrb

ihppEcALpeb

Ets4roEttcop

4

T

utation allows 4OHT to bind without disturbing H12 in ER�. ERR�-4OHTesidues shown in lavender with ligand in green and ER�-4OHT residues inlue and with ligand in orange. ERR� numbering was used in the figure.

n a different space group confirms his analysis. On the otherand, the ER�-4OHT structure has H12 adopting a modifiedosition upon 4OHT binding [44] binding at the co-activatorosition. The wider cavity of ER� with a leucine (L525) inRR�’s F435 position (Fig. 7) leaves enough space in theavity for 4OHT binding and shows no collisions with H12.lso, ER�’s H12 contains the LXXLL motif, with L536 and540 responsible for anchoring the peptide in the co-activatorosition. This motif is not present in ERR�’s H12. This couldxplain the differences in positioning of H12 upon antagonistinding between ERR� and ER�.

An overlay of the 4OHT molecules from Greischick’sRR�-4OHT structure, ER�-4OHT and our ERR� struc-

ure shows a conserved orientation between the three withome variation in the orientation of the amino group of theOHT molecule [31,44]. The structure of ER� with 4OHTeveals H12 packing against H3 and H4 occupying the placef the activation peptide according to the ERR�-SRC andRR�-PGC-1-a peptide structures [37,43,44]. These struc-

ures and its secondary structure rearrangement demonstratehat antagonist binding induces the rearrangement of the H12ausing a transcriptionally inactive protein. This flexibilitybserved in H12 gives us an indication of its ability to regulaterotein activity.

. Discussion

ERR� is a constitutively active orphan nuclear receptor.he molecular basis of its constitutive activity has been linked

emistry

totsouTothoiadtbtlts

othonistdtwcraoadsrrtetioaon

TrSilb

cTsac

R

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M.C. Abad et al. / Journal of Steroid Bioch

o the favorable conformation of H12, which allows bindingf co-activators even in the absence of ligands, as seen inhe crystal structure solved by Renaud et al. [37]. The tran-criptional activity of ERR� is tissue selective and dependsn promoter context and co-regulator recruitment. No nat-ral ligand have been identified that regulates its activity.hermoFluor® was used to screen the ligand-binding domainf the receptor and identified several classes of compoundshat stabilized the receptor. Traditional assays in nuclearormone receptor assays rely on competitive displacementf known ligands or on modulating receptor co-regulatornteraction. ThermoFluor® allows the identification of bothntagonists and functionally silent ligands, because it is airect binding assay. The latter class of ligands did not resulto further enhancement of the receptor for co-activator affinityut enhanced the thermodynamically stability of the recep-or. Superpositioning of the co-activator peptide in the silentigand co-crystal structures showed that it was possible forhe co-activator to bind, further supporting the functionallyilent response of these ligands.

Several reports support that the ERR’s are key mediatorsf the biological response for PGC-1 and RIP-140 on generanscription [23,24] and more recently additional reportsave shown that ERR’s are modulators of the monoaminexidase promoter, implicating them also as regulators of theeurotransmitter pathway [29,45]. The latter is of particularnterest considering that our functionally silent ligands aremall molecular weight phenols. It is tempting to speculatehat small catecholamine metabolites of the monoamine oxi-ase catabolic pathway can be putative ligands for ERR�. Weested several catecholamines in ThermoFluor® and althoughe observed no further stabilization (data not shown), one

annot rule out the existence of putative metabolites. Nucleareceptor ligands historically are assigned as antagonist orgonist based on their functional response in biochemicalr cellular assays. To date, they are no examples how willfunctional silent ligand that stabilizes the ligand-binding

omain of the receptor will manifest itself in vivo. One pos-ible mechanism is by thermodynamic stabilization of theeceptor we could alter intracellular steady state levels of theeceptor which can impact biological activity through cross-alk to other nuclear receptors [46], alter affinity for responselements [47] or specific co-regulator interactions [48] eventshat were not explored in this report. In the case of ERR� sincet is regarded constitutive active, by increasing steady levelsf the receptor the biological response of such a ligand willppear as an agonist. It remains to be seen if this new classf nuclear hormone ligands have the potential in becomingovel therapeutics.

In conclusion, we have demonstrated the utility ofhermoFluor® in identifying ligands for orphan nuclear

eceptors that are independent of their functional response.

pecifically we have shown how the technology was utilized

n the discovery of functionally silent ligands that stabi-ize ERR� and used crystallography to define the molecularasis of the silent response of BPA and ClCH3Ph in our

[

& Molecular Biology 108 (2008) 44–54 53

ellular trans-activation and co-activator TR-FRET assays.hese ERR� reported structures will assist in the design ofelective ERR� modulators using these ligands as seeds infragment based approach and will be discussed in future

ommunications.

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