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Originally published In Press as doi:10.1074/jbc.M402195200 on May 24, 2004

J. Biol. Chem., Vol. 279, Issue 32, 33639-33646, August 6, 2004
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Structural Basis for the Deactivation of the Estrogen-related Receptor {gamma} by Diethylstilbestrol or 4-Hydroxytamoxifen and Determinants of Selectivity*

Holger Greschik, Ralf Flaig, Jean-Paul Renaud, and Dino Moras{ddagger}

From the Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, B. P. 10142, 67404 Illkirch, France

Received for publication, February 27, 2004 , and in revised form, May 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen-related receptor (ERR) {gamma} behaves as a constitutive activator of transcription. Although no natural ligand is known, ERR{gamma} is deactivated by the estrogen receptor (ER) agonist diethylstilbestrol and the selective ER modulator 4-hydroxytamoxifen but does not significantly respond to estradiol or raloxifene. Here we report the crystal structures of the ERR{gamma} ligand binding domain (LBD) complexed with diethylstilbestrol or 4-hydroxytamoxifen. Antagonist binding to ERR{gamma} results in a rotation of the side chain of Phe-435 that partially fills the cavity of the apoLBD. The new rotamer of Phe-435 displaces the "activation helix" (helix 12) from the agonist position observed in the absence of ligand. In contrast to the complexes of the ER{alpha} LBD with 4-hydroxytamoxifen or raloxifene, helix 12 of antagonist-bound ERR{gamma} does not occupy the coactivator groove but appears to be completely dissociated from the LBD body. Comparison of the ligand-bound LBDs of ERR{gamma} and ER{alpha} reveals small but significant differences in the architecture of the ligand binding pockets that result in a slightly shifted binding position of diethylstilbestrol and a small rotation of 4-hydroxytamoxifen in the cavity of ERR{gamma} relative to ER{alpha}. Our results provide detailed molecular insight into the conformational changes occurring upon binding of synthetic antagonists to the constitutive orphan receptor ERR{gamma} and reveal structural differences with ERs that explain why ERR{gamma} does not bind estradiol or raloxifene and will help to design new selective antagonists.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen-related receptors ERR{alpha},1 ERR{beta}, and ERR{gamma} (NR3B1, -2, and -3) (1) form a subfamily of orphan nuclear receptors that share significant amino acid homology with the estrogen receptors ER{alpha} and ER{beta} (NR3A1 and -2) (2, 3). Because of the high conservation in the DNA binding domain, ERRs and ERs have overlapping DNA binding selectivity (4-6) and, accordingly, may co-regulate target genes in tissues in which they are co-expressed. ERR subfamily members have for example been shown to modulate the expression of ER target genes in bone (7, 8) or breast tissue (9, 10). Importantly, overexpression of ERR{alpha} and ERR{gamma} in samples from breast cancer patients correlates with unfavorable and favorable biomarkers, respectively (11). Therefore, these receptors might serve as prognostic markers themselves or even be targets for endocrine therapy in human breast cancer.

Despite their significant homology with ERs in the ligand binding domain (LBD), ERRs do not (or only very weakly) respond to estradiol (E2) (2, 12). Furthermore, whereas ERs are ligand-activated receptors, ERRs are constitutively active (13-16), and a structural study confirmed that the ERR{gamma} LBD can adopt a transcriptionally active conformation and interact with the steroid receptor coactivator 1 (SRC-1) in the absence of any ligand (12). Together, these observations suggest that ERRs are ligand-independent activators of transcription whose activation potential may rather be determined by the presence of transcriptional coactivators (17-20).

Although natural ERR agonists may not exist, putative endogenous ligands were predicted to act as antagonists due to the specific architecture of the ligand binding pocket (LBP) observed for the ERR{gamma} apoLBD (12). Although no natural ligand is known to date, the ER agonist diethylstilbestrol (DES) and the selective ER modulator (SERM) 4-hydroxytamoxifen (4-OHT) have been identified as ERR antagonists (21-23). DES deactivates all three isotypes, whereas 4-OHT binds only to ERR{beta} and ERR{gamma}. In contrast, the SERM raloxifene (RAL) does not bind to ERRs (23) (summarized in Table I). The activity of ERR{alpha} is also antagonized by the organochlorine pesticides chlordane and toxaphene (24), but binding of these substances to ERR{alpha} remains unclear (21). In contradiction to structure-based predictions (12), a recent study identified flavone and isoflavone phytoestrogens as ERR agonists (25), although binding of these substances to the receptors has not been demonstrated.


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  		TABLE I
Effect of selected ligands on the AF2 of ERs and ERRs

A dash indicates that the respective ligand does not or only very weakly affects the transcriptional activity of the nuclear receptor.

 
Nuclear receptor LBDs adopt a canonical antiparallel {alpha}-helical sandwich fold generally composed of 12 {alpha}-helices (H1-H12) and a small {beta}-sheet (26, 27). Agonist ligands trigger the LBD activation function (termed AF2) by stabilizing a defined conformation in which H12 packs against the LBD body and together with H3 and H4 generates a hydrophobic coactivator binding surface. Coactivators typically bind via LXXLL sequence motifs (L denotes leucine, and X denotes any amino acid) that form amphiphatic {alpha}-helices. Antagonist ligands interfere with the formation of an active LBD conformation and coactivator recruitment. In most documented cases AF2 antagonism is based on steric interference between a bulky ligand extension protruding from the LBP and H12 in the agonist position, as exemplified by the ER{alpha} LBD bound to RAL or 4-OHT (28, 29) or the ER{beta} LBD complexed with ICI 164,384 (ICI) (30). Alternatively, "small" antagonists without bulky extension can destabilize the active LBD conformation by promoting nonproductive interactions between H12 and the LBD body, as exemplified by the action of 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (R,R enantiomer) on ER{beta} (31).

In crystallographic and recent fluorescence studies it has been attempted to more precisely correlate the conformation of the antagonist-bound ER LBD with the distinct biological activities elicited by SERMs (RAL, 4-OHT) or full ER antagonists (ICI compounds) in vivo (28-30, 32). In the crystallized complexes of the ER{alpha} LBD with RAL or 4-OHT a partial unfolding of the C-terminal end of H11 and structural adaptations of the H11/H12 loop were observed, allowing H12 to bind to the coactivator groove (28, 29). In contrast, in the ER{beta} LBD·ICI complex H12 is not visible due to high mobility, since the terminal amide moiety of the ligand sterically precludes its packing against the coactivator groove (30). In turn, a high H12 mobility appears to result in an increased cellular ER turnover, accounting in part for the full antagonist activity of ICI compounds (30, 33). Fluorescence studies confirmed a partial unfolding of the C terminus of H11 in ER{alpha} LBD-antagonist complexes in solution (32). Interestingly, full antagonists seem to have a lower H11 unfolding potential than SERMs, and subtle differences were observed between the spectroscopic signatures of RAL and 4-OHT.

In contrast to ERs, nothing is known about ligand-induced conformational changes of ERR LBDs. Here we report the crystal structures of the ERR{gamma} LBD·DES complex (at 2.1 Å resolution) and of two ERR{gamma} LBD·4-OHT complexes (at 2.2 and 3.2 Å resolution). Superimposition of the antagonist-bound structures with the ERR{gamma} apoLBD (refined to 2.4 Å resolution) in the transcriptionally active conformation establishes the conformational changes occurring upon DES or 4-OHT binding. Comparison of the ERR{gamma} LBD-antagonist complexes with the ER{alpha} LBD bound to various ligands (E2, DES, 4-OHT, RAL) reveals differences in the architecture of the LBPs of ER{alpha} and ERR{gamma} that do not permit efficient binding of E2 or RAL to ERR{gamma}. Finally, our results suggest that (unlike observed in the complexes of the ER{alpha} LBD with RAL and 4-OHT) a highly mobile H12 interferes with the recruitment of coactivators to antagonist-bound ERR{gamma}.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Production and Purification—The hexahistidine-tagged murine ERR{gamma} LBD (residues 229-458) was produced from a pET-15b expression vector (Novagen) in Escherichia coli BL21(DE3) at 37 °C. After sonication of the bacterial pellet and ultracentrifugation, the recombinant protein was first purified by cobalt affinity chromatography (TALON, Clontech). Affinity chromatography was carried out in a batch at room temperature in buffer A (20 mM Tris-HCl (pH 8.0), 50 mM NaCl). After 3 washes of the TALON resin with buffer A containing 5 mM imidazole (pH 8.0), the hexahistidine-tagged ERR{gamma} LBD was eluted by increasing the imidazole concentration to 200 mM. The protein was further purified by gel filtration using a HiLoad 16/60 Superdex 200 column (Amersham Biosciences) on a BioLogic work station (Bio-Rad). The LBD homodimer eluted in buffer A from the gel filtration column at the expected position as a single peak. The fractions containing purified protein (estimated purity >95%) were pooled and concentrated to about 8 mg/ml.

Protein Complex Crystallization, Data Collection, and Processing—Co-crystallization with a 2-fold molar excess of DES or 4-OHT (Sigma) was carried out with the hanging drop or the sitting drop vapor diffusion method (hanging drop: 2 µl of protein solution plus 2 µl of reservoir against 500 µl of reservoir in 24-well VDX plates (Hampton Research); sitting drop: 1 µl of protein solution plus 1 µl of reservoir against 200 µl of reservoir in 96-well CrystalQuickTM plates (Greiner)). For the ERR{gamma} LBD·DES and one of the ERR{gamma} LBD·4-OHT complexes, the PEG/Ion Screen (Hampton Research) allowed identification of preliminary crystallization conditions. A second crystal form for the ERR{gamma} LBD·4-OHT complex was found upon screening with the Crystal Screen II (Hampton Research). In the refined conditions, ERR{gamma} LBD·DES crystals grew in sitting drops within a few days at 24 °C with a reservoir containing 0.1 M Tris-HCl (pH 8.0), 0.1 M sodium acetate, 18% PEG3350 (cryo conditions: 0.1 M Tris-HCl (pH 8.0), 0.1 M sodium acetate, 20% PEG3350, 20% ethylene glycol). ERR{gamma} LBD·4-OHT crystals (crystal form 1) were obtained in hanging drops at 4 °C with a reservoir containing 0.1 M MES (pH 6.5), 1.6 M MgSO4 (cryo conditions: 0.1 M MES (pH 6.5), 1.6 M MgSO4, 13% ethylene glycol). ERR{gamma} LBD·4-OHT crystals of form 2 grew in hanging drops at 24 °C in reservoir containing 0.1 M Tris-HCl (pH 8.0), 0.2 M MgSO4, 14% PEG3350 (cryo conditions: 0.1 M Tris-HCl (pH 8.0), 0.1 M MgSO4, 20% PEG3350, 20% ethylene glycol). New crystals of the ERR{gamma} apoLBD grew in the presence of a 3-fold molar excess of a SRC-1 coactivator peptide (686RHKILHRLLQEGSPS700) (12) at 4 °C in hanging drops with a reservoir containing 100 mM Tris-HCl (pH 8.5), 1.6 M (NH4)2SO4, 20% glycerol (the mother liquor served as cryoprotectant). X-ray diffraction data were collected at the ID14-1, the ID14-2, or the BM30 beam line at the European Synchrotron Radiation Facility in Grenoble, France. The data were integrated and scaled using the HKL2000 package (34).

Structure Determination, Refinement, and Comparison—The crystal structures of the ERR{gamma} LBD·DES and the ERR{gamma} LBD·4-OHT complexes were solved by molecular replacement with AMoRe (35) using the C-terminal-deleted ERR{gamma} LBD homodimer (amino acids 235-440) (12) as the search model. The structures were refined at the indicated resolution (Table II) using CNS (36) and programs of the CCP4 package (37-40). Manual adjustments and rebuilding of the models were performed using the program O (41). The final models were validated with PROCHECK (42). Data collection and structure refinement statistics are summarized in Table II. For structure comparisons, the C{alpha} traces of the models were superimposed from H3 (Leu-265 in ERR{gamma}; Met-343 in ER{alpha}) to the middle of H11 (Phe-435 in ERR{gamma}; Leu-525 in ER{alpha}) using the lsq commands of O and default parameters. The probe-occupied volume of the ERR{gamma} LBP in the absence and presence of ligand was calculated with VOIDOO (43). The figures were generated with DINO.2


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  		TABLE II
Data collection and refinement statistics

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure of the Antagonist-bound ERR{gamma} LBD—We solved the crystal structures of the ERR{gamma} LBD in complex with DES (at 2.1 Å of resolution, space group P21) and with 4-OHT (crystal form 1 at 2.2 Å resolution, space group P4322; crystal form 2 at 3.2 Å resolution, space group P3221) (Table II). In addition, we further refined the structure of the ERR{gamma} apoLBD bound to a SRC-1 peptide (12) at 2.4 Å of resolution (space group P43212). In all cases, the ERR{gamma} LBD crystallized in homodimeric form; thus, ligand binding does not interfere with homodimer formation.

Comparison of the ERR{gamma} apoLBD with the DES and 4-OHT complexes revealed no major conformational changes for the main chain atoms from H1 to the middle of H11 (data not shown). Some changes of the main chain include adaptations of the H1-H3 loop, which mainly result from crystal packing. In contrast, important structural perturbations of the main chain due to 4-OHT or DES binding affect the C terminus of H11, the H11/H12 loop, and H12 (Table III). First, in the ligand-bound complexes, the C terminus of H11 is slightly bent away from the entrance of the LBP due to space requirements of the ligand. Second, in some subunits of the ERR{gamma} LBD·4-OHT (but not the ERR{gamma} LBD·DES) complexes present in the asymmetric unit (AU) the C-terminal end of H11 is unfolded. In contrast to ER{alpha} LBD·SERM complexes (28, 29), partial H11 unfolding is not consistently observed in the ERR{gamma} LBD·4-OHT complexes but is only found in one subunit in both crystal forms. Finally, in all ligand-bound complexes the H12 region adopts distinct conformations or is found in different positions. The H11/H12 loop adapts to the constraints imposed by the exact position and conformation of H11 and H12 (Table III).


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  		TABLE III
Folding of the H11-H12 region

 
In contrast to the complexes of the ER{alpha} LBD with RAL and 4-OHT (28, 29), in none of the ERR{gamma} LBD-antagonist complexes does H12 pack against the coactivator groove of its respective LBD body. H12 is not visible, most likely due to high mobility in the case of the ERR{gamma} LBD·4-OHT complex (crystal form 2), and interacts with the coactivator groove of a neighboring molecule in crystal form 1. For subunits A and B, found in the AU of crystal form 1, this interaction requires different conformational adaptations of the H11/H12 loop and the N terminus of the H12 region (Fig. 1A). In the ERR{gamma} LBD·DES complex, the H12 region is not visible or is unfolded in three of four molecules (subunits A-C). H12 of the fourth molecule (subunit D) is stabilized through crystal contacts in a position that resembles the agonist conformation found in the ERR{gamma} apoLBD·SRC-1 complex (Fig. 1B). However, in this "agonist-like" position, H12 of subunit D is shifted by one residue along the {alpha}-helical axis with respect to the agonist H12 position in the ERR{gamma} apoLBD·SRC-1 complex. As a consequence the conformation of the H11/H12 loop is completely changed, and H12 occupies a position that in solution is most likely not stabilized by favorable contacts. Together, these observations suggest that in solution DES or 4-OHT binding strongly increases the mobility of H12, whose conformation and position in the crystals is apparently determined by packing interactions.



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  		FIG. 1.
H12 positions found in complexes of the ERR{gamma} LBD with 4-OHT or DES. A, superimposition of subunits A (orange) and B (black) present in the AU of the ERR{gamma} LBD·4-OHT complex (crystal form 1). In both cases H12 packs against the coactivator groove of a neighboring molecule with distinct adaptations of the H11/H12 region. Amino acids 443 and 444 of subunit A and 439-444 of subunit B are not well defined by electron density and have been omitted from the final model. B, shifted agonist-like position of H12 in subunit D of the ERR{gamma} LBD·DES complex (green). The corresponding parts of the ERR{gamma} apoLBD (yellow) with H12 in the transcriptionally active position have been superimposed. In the DES complex the conformation of the H11/H12 loop and the position of H12 are determined by crystal packing and are most likely of no physiological relevance.

 
Mechanism of DES- and 4-OHT-mediated Antagonism—Because of size limitations, DES or 4-OHT cannot bind inside the small ERR{gamma} LBP (~220 Å3) observed in the apoLBD (12). Consequently, DES binding induces structural adaptations that enlarge the volume of the ERR{gamma} LBP to about 330 Å3. The 4-OHT binding niche differs from the DES cavity mainly around the amine moiety, which extends onto the protein surface. The most prominent conformational change inside the LBP in response to DES and 4-OHT binding is a rotation of the side chain of Phe-435 (H11), which mostly accounts for the increase in cavity volume (Fig. 2). The new rotamer of Phe-435 would sterically clash with H12 in the agonist position, notably with the side chain of Leu-454 in H12 (Fig. 2A). Thus, the DES- or 4-OHT-induced rotation of the side chain of Phe-435 precludes formation of the canonical transcriptionally active conformation of the ERR{gamma} LBD, which consequently impairs coactivator recruitment.



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  		FIG. 2.
Conformational changes inside the ERR{gamma} LBP upon antagonist binding. A, superimposition of selected parts of the ERR{gamma} LBD·DES complex (green) with the ERR{gamma} apoLBD (yellow). For the apoLBD, H12 in the agonist position has been included. The largest side chain movements in response to DES binding are observed for Phe-435 and Glu-275, whereas Tyr-326, Asn-346, and His-434 adapt to a lesser extent. The rotation of the side chain of Phe-435 upon DES binding results in steric interference with H12 in the agonist position. B, superimposition of selected parts of the ERR{gamma} LBD complexed with 4-OHT (orange) or DES (green). The ligands occupy slightly different positions inside the ERR{gamma} cavity. C, DES (left) or 4-OHT (right) bound to the ERR{gamma} LBD fitted into 2Fo - Fc electron density maps.

 
The binding niche of DES or 4-OHT is delineated by amino acid residues from H3 (Leu-265, Leu-268, Cys-269, Leu-271, Ala-272, Glu-275), H5 (Trp-305, Met-306, Leu-309, Ile-310, Val-313, Arg-316), the {beta}-sheet (Tyr-326), H7 (Leu-342, Leu-345, Asn346, Ile-349), and H11 (Ala-431, His-434, Phe-435) (Fig. 2 and data not shown). The "anti-estrogenic" amine moiety of 4-OHT forms additional contacts with Trp-305 and Phe-435 (data not shown). Furthermore, the side chain of Asp-273 (H3) on the ERR{gamma} LBD surface reorients to form a hydrogen bond with the tertiary amine group of 4-OHT (Fig. 2B), which has also been observed with Asp-351 in the ER{alpha} LBD·4-OHT complex (see Fig. 3B) (29). As in the corresponding ER{alpha} complexes (29), DES and 4-OHT are anchored on one side of the LBP by hydrogen bonds with Glu-275 (H3) and Arg-316 (H5), while DES forms another hydrogen bond with His-434 (H11) on the other side of the cavity. Importantly, the side chain of Glu-275, whose position is not well defined in the ERR{gamma} apoLBD due to high mobility, reorients toward the ligand and is tightly positioned in the DES and the 4-OHT complexes. In comparison, Arg-316 and His-434 only slightly reorient upon ligand binding. Other small structural changes inside the LBP relative to the apoLBD concern the side chains of Tyr-326 ({beta}-sheet) and Asn-346 (H7) interacting through a hydrogen bond, which adapt to space requirements of the ligand. Superimposition of the DES and 4-OHT complexes of the ERR{gamma} LBD shows that neither the aromatic A ring nor the A ring of DES superimpose exactly with the corresponding A or B ring of 4-OHT (Fig. 2B). This observation reveals a small degree of variability, by which different types of ligands can use the available space inside the ERR{gamma} LBP.



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  		FIG. 3.
Comparison of the LBPs of ERR{gamma} and ER{alpha}. A, superimposition of selected parts of the ERR{gamma} LBD·DES complex (green) with the ER{alpha} LBD·DES complex (red) (PDB code 3ERD [PDB] ). Although the overall ligand binding mode is very similar in both complexes, the DES binding position is shifted by about 0.5 Å in the cavity of ERR{gamma} relative to ER{alpha}. In contrast to ERR{gamma}, H12 of the ER{alpha} LBD can occupy the agonist position since there is no steric clash with Leu-525 (corresponding to Phe-435 in ERR{gamma}). B, superimposition of selected parts of the ERR{gamma} LBD·4-OHT complex (orange) with the ER{alpha} LBD·4-OHT complex (blue) (PDB code 3ERT [PDB] ). In ERR{gamma}, the 4-OHT binding position is slightly rotated and mainly differs around the B ring and the amine moiety. The altered 4-OHT binding results from steric constraints imposed by Leu-345 (Ile-424 in ER{alpha}), a slightly shifted relative position of H7, and the side chain of Phe-435 (Leu-525 in ER{alpha}). C, left, superimposition of 4-OHT (blue), E2 (light gray), and RAL (black) as observed in the LBP of the respective ER{alpha} complexes (PDB codes: ER{alpha}/E2, 1ERE [PDB] ; ER{alpha}/RAL, 1ERR [PDB] ). Right, superimposition of 4-OHT as observed in the cavity of ERR{gamma} (orange) with E2 (light gray) and RAL (brown) bound to ER{alpha}. E2 and RAL protrude deeper into the cavity of ER{alpha} than 4-OHT and do not bind to ERR{gamma} due to insufficient space in the LBP.

 
Comparison with ER{alpha} and Determinants of Ligand Binding Selectivity—Structure superimposition reveals an overall similar mode of DES binding to ERR{gamma} and ER{alpha}, the major principal difference between the complexes being a canonical agonist H12 position in the case of ER{alpha} but various antagonist H12 conformations in the case of ERR{gamma} (Fig. 3A). DES binding does not interfere with the agonist H12 position in ER{alpha} because the side chain of Leu-525 (corresponding to Phe-435 in ERR{gamma}) leaves enough space to accommodate the ligand without a large conformational change. Interestingly, the binding position of DES is slightly shifted by about 0.5 Å in the ERR{gamma} relative to the ER{alpha} cavity. This shift could result from differences in the relative positions of individual {alpha}-helices and, thus, the exact topology of the LBPs. Of the secondary structure elements that contribute to the cavity, H3, H5, and the {beta}-sheet superimpose well with only minor positional deviations. In contrast, significant shifts of 1.0-1.5 Å along the helix axis are observed for H7 and H11 of ERR{gamma} (Fig. 3, A and B). Both shifts restrict the available space in the ERR{gamma} LBP, which may be the cause of the slightly altered DES binding position.

Importantly, compared with DES, the 4-OHT binding position differs more significantly in the LBPs of ER{alpha} and ERR{gamma} (Fig. 3B). Although the overall binding mode is conserved and the aromatic ligand A ring and surrounding parts of the LBP superimpose relatively well in both complexes, the 4-OHT binding positions differ for the B ring and the amine moiety. Steric constraints imposed by Leu-345 (H7) and Phe-435 (H11) in ERR{gamma} (corresponding to Ile-424 and Leu-525 in ER{alpha}, respectively) lead to a small rotation of the 4-OHT molecule around C1 in the aromatic A ring. A 4-OHT binding position in the LBP of ERR{gamma}, as observed in ER{alpha}, would result in too close contacts with Leu-345 (~3 Å) and Phe-435 (~2.8 Å).

These differences in the architecture of the LBPs of ERR{gamma} and ER{alpha} provide a good explanation why E2 and RAL do not bind to ERR{gamma} with significant affinity. Superimposition of ER{alpha} LBDs in complex with different ligands shows that E2 and RAL protrude deeper into the LBP toward Ile-424 than 4-OHT (Fig. 3, B and C), resulting in conformational adaptations including distinct side chain conformations of Ile-424 (H7) (28, 29). In contrast, it appears that despite the dynamic behavior of proteins, the space restrictions in the cavity of ERR{gamma} imposed by the shifted position of H7 cannot be compensated in an energetically favorable manner by a conformational change of the side chain of Leu-345. Thus, the altered position of H7 and the presence of Leu-345 in ERR{gamma} (replacing Ile-424 in ER{alpha}) seem to account mostly for the lack of E2 or RAL binding to this receptor.

In One of the ERR{gamma} LBD·4-OHT Complexes the Packing of H12 against the Coactivator Groove of a Neighboring Molecule Is Determined by a Fortuitously Co-crystallized Bile Acid Molecule—Finally, we compared the H12/coactivator groove interactions in the 4-OHT complexes of ERR{gamma} (crystal form 1) and ER{alpha}. In ER{alpha} H12 packs tightly against its respective LBD body and mimics coactivator binding by inserting Leu-536 and Leu-540 into the coactivator cleft (29). In contrast, in ERR{gamma} the interaction of H12 with the LBD body of a neighboring molecule is determined by the presence of a fortuitously co-crystallized molecule. During refinement the clearly contoured, additional electron density allowed us to identify this molecule as cholic acid (or a closely related bile acid) (Fig. 4A). The bile acid is bound close to the entrance of the LBP within the proximity of the protruding amine moiety of 4-OHT (shortest distance ~3.2 Å) and occupies together with the C-terminal portion of H12 the hydrophobic coactivator groove (Fig. 4B). Comparison of both ERR{gamma} subunits present within the AU shows that most regions including H3, cholic acid, and the C-terminal part of H12 superimpose well, whereas the H11/H12 loop and the N terminus of H12 adopt distinct folds due to different relative H12 positions (Fig. 1A and data not shown). In the middle of H12, Phe-450 of ERR{gamma} interacts with the coactivator cleft in a roughly similar manner as Leu-540, the corresponding residue in ER{alpha} (Fig. 4B). Packing of the N-terminal portion of the ERR{gamma} H12 against the coactivator cleft of a neighboring molecule, however, is precluded by the presence of the cholic acid molecule and determined by the distinct conformations of the H11/H12 loop in both ERR{gamma} subunits. Consequently, Met-446 of ERR{gamma} does not form an interaction equivalent to that of the corresponding Leu-536 in ER{alpha}.



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  		FIG. 4.
Interactions of H12 with the coactivator groove in the ER{alpha} LBD·4-OHT and the ERR{gamma} LBD·4-OHT complex (crystal form 1). A, fitting of the fortuitously co-crystallized cholic acid molecule into a 2Fo - Fc electron density map. B, superimposition of selected parts of the ERR{gamma} LBD (orange) and the ER{alpha} LBD (blue) bound to 4-OHT. In the ER{alpha} LBD·4-OHT complex, H12 packs against the coactivator cleft of the respective LBD body with Leu-536 and Leu-540, mimicking LXXLL coactivator interactions. In the ERR{gamma} LBD·4-OHT complex (subunit B in crystal form 1), H12 interacts with the coactivator groove of a neighboring molecule. Binding is distinct from that in ER{alpha} due to the absence of a LXXLL motif in H12 of ERR{gamma}. The alternative packing interactions between H12 and the coactivator groove, rather, are determined by the fortuitously co-crystallized cholic acid molecule that occupies part of the cleft.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Mechanism of ERR{gamma} Deactivation—In this study we compare the crystal structures of the ERR{gamma} LBD bound to the antagonists DES or 4-OHT with the apoLBD in the transcriptionally active conformation. Our comparisons establish that DES- and 4-OHT-mediated antagonism is based on the rotation of the side chain of Phe-435, which upon ligand binding flips out of the LBP and sterically interferes with H12 in the agonist position. As a consequence, H12 is dissociated from the LBD body, and coactivator binding is impaired. These ideas are in agreement with previous results showing that mutation of Phe-435 to leucine (the corresponding residue in ER) abolishes the antagonist action of DES on ERR{gamma} in functional assays (12). Because 4-OHT binding also requires a rotation of the side chain of Phe-435, the ligand amine moiety protruding from the LBP is not primarily responsible for the antagonist action of 4-OHT on ERR{gamma}.

Antagonist Ligand Effects on ERR{gamma} and ERs—AF2 antagonists of ERs have been classified as "active" if a bulky ligand extension interferes with the agonist H12 position (RAL, 4-OHT, ICI) and as "passive" in the case of small ligands (5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol) (28-31). Deactivation of ERR{gamma} by DES (a ligand without bulky extension) provides a particular case of "active" antagonism, in which a small ligand acts as antagonist by inducing the rotation of the side chain of a single residue (Phe-435) inside the LBP.

In an attempt to correlate antagonist-induced conformational changes of the ER LBD with ligand activities in vivo, AF2 antagonists with bulky extension have been further sub-classified according to their H11 "unfolding potential" and their potential to increase the mobility of H12 or to promote H12 packing against the coactivator cleft (28-30, 32). Although most observations from structural and fluorescent studies appear consistent, questions remain about the dynamic behavior of H12 in solution, since the packing of H12 against the coactivator cleft observed in the crystal structures of the ER{alpha} LBD with RAL and 4-OHT may be due to the crystallization conditions. In fact, to our knowledge the dynamic behavior of H12 of ER in the presence of SERMs or full antagonists has never been comparatively analyzed in fluorescence anisotropy experiments as previously applied for the wild-type or mutant peroxisome proliferator-activated receptor {gamma} LBD (45).

Similarly, the effects of ligand binding on the conformation of the ERR{gamma} LBD and the dynamic behavior of H12 have not been addressed in fluorescence studies. In the absence of such data, the presented crystal structures allow first insight into the conformational changes induced by DES and 4-OHT. Most parts of the ERR{gamma} LBD (H1 to the middle of H11) do not undergo significant conformational changes upon antagonist binding. Structural adaptations affect the C-terminal part of H11, which slightly bends away from the entrance of the LBP due to space requirements of the ligand but only unfolds in individual subunits within the AU of the ERR{gamma} LBD·4-OHT complexes. Interestingly, DES and 4-OHT may have distinct unfolding potentials, since we do not observe partial H11 unfolding in the DES complexes. Binding of both antagonists, however, dissociates the H12 region from the LBD body. The H12 positions and conformations observed in the different complexes seem to be random and dictated by crystallization conditions or crystal packing rather than correlate with ligand characteristics. Notably, unlike that observed for the ER{alpha} LBD·4-OHT complex (29), H12 does not pack against the coactivator cleft of its respective LBD body in the ERR{gamma} LBD·4-OHT complexes. Because the length of the H11/H12 region is identical in both receptors, differences in the amino acid sequence may explain this observation. H12 of ER{alpha} contains an LXXLL "coactivator" motif, of which Leu-536 and Leu-540 mediate the anchoring of H12 in the coactivator cleft in the RAL and the 4-OHT complex (28, 29). In contrast, no LXXLL motif is present in H12 of ERR{gamma}, and the corresponding residues (Met-446 and Phe-450, respectively) may not allow efficient interactions of H12 with the coactivator groove. Therefore, the dissociation of H12 from the LBD body upon DES or 4-OHT binding to ERR{gamma} rather resembles the action of the fully antagonistic ICI compounds on ERs. However, the in vivo consequences of antagonist binding to ERR{gamma} (e.g. an enhanced receptor degradation as for ER/ICI complexes (30, 33) or potential tissue-specific effects as for ER{alpha}·SERM complexes (46)) remain to be established in future studies.

A "Fortuitous" Cholic Acid Molecule Bound at the Surface of the ERR{gamma} LBD—Unexpectedly, in crystal form 1 of the ERR{gamma} LBD·4-OHT complex, cholic acid (or a closely related bile acid) was co-crystallized. One possible explanation for the presence of cholic acid is that the molecule has been co-purified from the E. coli expression host, since bacteria present in the human colon are known to be involved in the metabolism of bile acids that escape the enterohepatic circulation (between the liver, gallbladder, and intestines) (47). Cholic acid together with parts of H12 from a neighboring molecule covers the hydrophobic coactivator groove and, thus, stabilizes the crystal packing, a phenomenon that has been previously observed for a detergent molecule shielding the hydrophobic cleft in crystals of the RAR{gamma} LBD (48). The proximity of cholic acid to the entrance of the LBP and the amine moiety of 4-OHT raises the possibility that ligands may be designed that further extend onto the ERR{gamma} LBD surface. However, such an approach will require a more careful analysis of the "binding" mode of cholic acid.

Currently, we do not see any physiological relevance for the observed association of cholic acid with the ERR{gamma} LBD. Although ERR{gamma} seems to be expressed at low levels in the small intestine and the liver (13, 49-51), the binding position of the bile acid at the ERR{gamma} surface is clearly determined by the crystal packing and only compatible with an antagonist but not an agonist LBD conformation. It, therefore, also remains unclear how cholic acid was "fortuitously" co-purified with the ERR{gamma} apoLBD.

Ligand Binding Selectivity of ERRs—ERRs have been shown to bind DES (all ERRs) and 4-OHT (ERR{beta} and ERR{gamma}) but not E2 or RAL (21-23). The comparison of the DES and 4-OHT complexes of the ERR{gamma} and the ER{alpha} LBDs reveals small but significant differences in the architecture of the LBP that explain ligand binding selectivity. The slightly shifted binding position of DES and the small rotation of 4-OHT in the LBP of ERR{gamma} apparently result from space restrictions due to the presence of Leu-345 (Ile-424 in ER{alpha}) and, relative to ER{alpha}, a shifted position of H7. Furthermore, the rotated side chain of Phe-435 (Leu-525 in ER{alpha}) "pushes" the 4-OHT amine moiety toward Asp-273. Importantly, in ER{alpha} Ile-424 can adapt through conformational changes to ligands such as E2 or RAL that protrude much deeper into the cavity than 4-OHT (28, 29). In ERR{gamma}, however, even taking into account protein dynamics, equivalent conformational adaptations of Leu-345 apparently cannot occur in an energetically favorable manner, thus precluding E2 or RAL binding. These ideas are supported by previous results showing significant binding of E2 to the mutants ERR{gamma} LBD(L345I) and ERR{gamma} LBD(L345I/F435L) in nondenaturing mass spectrometry analysis (12). Together, our observations suggest that synthetic estrogens, which as RAL deeply protrude into the ER LBP toward Ile-424, are unlikely to bind to ERR{gamma} (or other ERR isotypes), whereas synthetic estrogens like 4-OHT, which do not completely fill the space around Ile-424, are also potential ERR ligands.

In a recent study Suetsugi et al. (25) identified certain flavone and isoflavone phytoestrogens as ERR agonists, albeit without demonstrating their binding to ERR LBDs. The authors used a model of the ERR{alpha} LBD based on the agonist-bound ER{alpha} LBD to predict the presence of a LBP that can accommodate flavone or isoflavone ligands without interfering with the agonist conformation. Our superimposition of the antagonist-bound ERR{gamma} LBDs with the ER{beta} LBD-genistein complex (52) indicates a possible steric interference between the side chain of Leu-345 of ERR{gamma} and O4 (~2.2 Å), C3 (~3 Å), and C4 (~2.9 Å) of genistein (data not shown). In comparison, small adaptations of the LBP might permit the binding of daidzein, which lacks the hydroxyl group on C4 of genistein. However, superimposition with the ERR{gamma} apoLBD clearly predicts daidzein or related molecules to act like DES and 4-OHT as antagonists. Because our models of the ERR{alpha} and the ERR{beta} LBD, which are based on the ERR{gamma} rather than the more distantly related ER{alpha} LBD, suggest an overall very similar architecture of the LBPs (data not shown), our prediction is valid for all ERR isotypes. Structure determination of the apo and ligand-bound LBD of ERR{alpha} and ERR{beta} will eventually clarify this issue.

Assuming that the overall architecture of the LBP is conserved among ERRs, the ERR{gamma} LBD crystal structures provide a good starting point to attempt the design of isotype-selective antagonists. An important discriminative feature between the LBPs of ERR{beta} and ERR{gamma} are Tyr-326 ({beta}-sheet) and Asn-346 (H7), which form a hydrogen bond in ERR{gamma}. In ERR{beta} these residues correspond to Tyr-301 and Tyr-321, respectively. The different size and hydrogen-bonding properties of Asn-346 (ERR{gamma}) and Tyr-321 (ERR{beta}) could be essential in the design of ERR{beta}- or ERR{gamma}-selective ligands. In ERR{alpha} the corresponding residues are Phe-382 and Gly-402, respectively. Especially, the small residue Gly402 may allow modification of ligands such that the additionally available space is filled, possibly resulting in higher affinity and ERR{alpha} selectivity.

Our ERR{alpha} LBD model suggests that the receptor does not bind 4-OHT due to the side chain of Phe-328 (H3), corresponding to Ala-247 in ERR{beta} and Ala-272 in ERR{gamma} (12). Phe-328 (H3) and Phe-495 (H11; corresponding to Phe-435 in ERR{gamma}) are predicted to almost completely fill the ERR{alpha} LBP. In agreement with this model, ERR{alpha} binds 4-OHT upon mutation of Phe-328 to alanine (23), whereas an ERR{gamma} (A272F) mutant no longer responds to 4-OHT (12). Because the ERR{alpha} cavity appears to be filled, dramatic conformational changes may occur upon the binding of DES. This idea is interesting with respect to the recently published LBD crystal structures of Nurr1 (53) and DHR38 (the Drosophila homologue of NGFI-B) (54), in which the LBP is completely filled with four aromatic residues conserved within the NGFI-B subfamily. The authors propose that these orphan receptors are unlikely to be ligand-regulated since no apparent conformational changes of the aromatic residues could open a cavity. However, given the dramatic conformational changes that may occur upon the binding of DES to ERR{alpha} and the high adaptability observed for the LBPs of PXR (55) and EcR (44), it is tempting to speculate that (synthetic) ligands might be identified for members of the NGFI-B subfamily.

In summary, the results presented in this study establish the conformational changes of the ERR{gamma} LBD occurring upon DES or 4-OHT binding and reveal determinants of selective ligand binding and differences in the dynamic behavior of H12 of ERRs versus ERs.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1TFC [PDB] (ERR{gamma} apoLBD (refined structure)), 1S9P [PDB] (ERR{gamma} LBD·DES), 1S9Q [PDB] (ERR{gamma} LBD·4-OHT (crystal form 1)), 1VJB [PDB] (ERR{gamma} LBD·4-OHT (crystal form 2)).) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by a Marie-Curie individual fellowship of the European Community (to H. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 33-3-88-65-32-20; Fax: 33-3-88-65-32-76; E-mail: moras{at}igbmc.u-strasbg.fr.

1 The abbreviations used are: ERR, estrogen-related receptor; ER, estrogen receptor; AF, activation function; AU, asymmetric unit; DES, diethylstilbestrol; E2, estradiol; H, {alpha}-helix; 4-OHT, 4-hydroxytamoxifen; ICI, ICI 164,384; LBD, ligand binding domain; LBP, ligand binding pocket; RAL, raloxifene; SERM, selective estrogen receptor modulator; SRC-1, steroid receptor coactivator-1; MES, 4-morpholine-ethanesulfonic acid; PEG, polyethylene glycol. Back

2 A. Philippsen (2002) DINO: visualizing structural biology; www.dino3d.org. Back


   ACKNOWLEDGMENTS
 
We thank André Mitschler and Gilbert Bey for help with data collection, Souphatta Sasorith and Jean-Marie Wurtz for providing models of the ERR{alpha} and the ERR{beta} LBD, Anastassis Perrakis for help with ARP/wARP, and Isabelle Billas for critical reading of the manuscript. We are grateful to Joanne McCarthy, Steffi Arzt, Stéphanie Monaco, Carlo Petosa, and Philippe Carpentier, our local contacts at the European Synchrotron Radiation Facility (Grenoble).



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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