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Originally published In Press as doi:10.1074/jbc.M611424200 on February 5, 2007

J. Biol. Chem., Vol. 282, Issue 14, 10449-10455, April 6, 2007
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Structural Insights into Corepressor Recognition by Antagonist-bound Estrogen Receptors*Formula

Nina Heldring, Supported by grants from the Swedish Research Council and the Swedish Cancer Society{ddagger}1, Tanya Pawson§, Donald McDonnell, Eckardt Treuter{ddagger}2, Jan-Åke Gustafsson{ddagger}3, and Ashley C. W. Pike, Supported by Wellcome Trust Career Development Award Grant 064803§4

From the {ddagger}Department of Biosciences and Nutrition, Karolinska Institutet, S-14157 Huddinge, Sweden, §Structural Biology Laboratory, University of York, York YO10 5YW, United Kingdom, and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, December 13, 2006 , and in revised form, February 1, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Direct recruitment of transcriptional corepressors to estrogen receptors (ER) is thought to contribute to the tissue-specific effects of clinically important ER antagonists. Here, we present the crystal structures of two affinity-selected peptides in complex with antagonist-bound ER{alpha} ligand-binding domain. Both peptides adopt helical conformations, bind along the activation function 2 coregulator interaction surface, and mimic corepressor (CoRNR) sequence motif binding. Peptide binding is weak in a wild-type context but significantly enhanced by removal of ER helix 12. This region contains a previously unrecognized CoRNR motif that is able to compete with corepressors for binding to activation function 2, thereby providing a structural explanation for the poor ability of ER to directly interact with classical corepressors. Furthermore, the ability of other sequence motifs to mimic corepressor binding raises the possibility that coregulators do not necessarily require CoRNR motifs for direct recruitment to antagonist-bound ER.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The biological significance of corepressor recruitment by nuclear receptors (NRs)5 in gene repression is well documented (reviewed in Ref. 1). Known NR corepressors have been isolated in complexes together with histone deacetylases, which facilitate gene repression through deacetylation of histone tails (2). Even though the components of several complexes have been identified, less is known about the processes controlling their recruitment by NRs. Unlike most NRs, the estrogen receptor (ER) is unusual in that it does not appear to be repressed in the absence of hormone; consequently, the importance of NR corepressors in ER-mediated transcriptional signaling remains controversial. However, a number of recent studies have demonstrated that both agonist- and antagonist-bound ERs are able to recruit a variety of proteins that can repress its activity (3). Differential coregulator recruitment is also known to contribute to the tissue-specific effects of selective ER modulators (SERMs), a therapeutically important class of ER ligands that exhibit characteristics of both estrogens and anti-estrogens depending on the tissue (4, 5).

Direct coactivator/corepressor recruitment by NRs is primarily mediated by the receptor's activation function 2 (AF2) located in the C-terminal ligand-binding domain (LBD) between helices H3, H5, and H12. Structural analyses have demonstrated that AF2 activity is dictated by the orientation of the mobile C-terminal AF2 activation helix (H12) (6). Agonist binding stabilizes an "active" orientation of H12 resulting in the formation of a specific binding site for the LXXLL interaction motifs of NR coactivators (7). NR antagonists prevent proper alignment of H12 and induce receptor conformations in which H12 is repositioned so that it occludes the AF2 binding site (7, 8), is completely dissociated from the body of the LBD (9), or is bound to another site outside the AF2 region (10). In the latter cases, the AF2 binding site is accessible and can interact with the extended LXXXIXXXL CoRNR consensus motifs of NR corepressors.

Affinity-selected peptides that recognize SERM-bound ERs have been isolated from both random (11, 12) and focused (13) peptide libraries. These studies have revealed a variety of hydrophobic sequence motifs that act as highly specific conformational probes and are good predictors of the biological effects of a particular ligand (14). Such motifs also provide information regarding potential ligand-specific ER-coregulator interaction sites (11, 15, 16). McDonnell and co-workers (12, 13) have reported the isolation and identification of a number of short peptides using phage display that specifically recognize 4-hydroxytamoxifen (OHT)-bound ER. This study focuses on two such peptides. The OHT-specific {alpha}betaV peptide (SPG-SREWFKDMLS) was isolated from a random peptide library and contains a novel interaction motif (12). A second peptide, bT1 (hereafter referred to as CoRNRER box; sequence DAFQL-RQLILRGLQDD) was isolated from a focused library based on the corepressor consensus motif (13).

To understand the structural basis for the interaction between ER- and SERM-specific motifs, we have crystallized and solved the structure of SERM-bound ER{alpha}LBD in complex with both an affinity-selected CoRNR box peptide and a tamoxifen (OHT)-specific peptide. This study extends previous structural information on corepressor binding to NRs and provides novel insights into the specific recognition of the antagonist-bound state of ER.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials— 4-Hydroxy-tamoxifen, 17beta-estradiol, and raloxifene were purchased from Sigma-Aldrich. Biotinylated and crystallization-grade peptides (>95% purity) were purchased from Thermo Electron (Ulm, Germany).

Constructs—Human ER{alpha} and ERbeta cDNA cloned into VP16 expression vector (Clontech) were used as templates for mutagenesis. Mutations were introduced using the QuikChange XL site-directed mutagenesis kit (Stratagene). Generation of the domain-deleted ER{alpha} and ERbeta constructs and the ER{alpha}D351Y, E542A, G442H, and E443A and ERbetaE448A have been described previously (11, 15). Introduction of amino acid changes was made by using the following primers: ER{alpha}I358R (sense primer 5'-GAGCTGGTTCACATGAGAAACTGGGCGAAGAGG and antisense 5'-CCTCTTCGCCCAGTTTCTCATGTGAACCAGCTC), ER{alpha}K362A (sense primer 5'-CATGATCAACTGGGCGGCGAGGGTGCCAGGCTTTGTGG and antisense 5'-CCACAAAGCCTGGCACCCTCGCCGCCCAGTTGATCATG), ER{alpha}L372A (sense primer 5'-CTTTGTGGATTTGACCCGCCATGATCAGGTCCAC and antisense 5'-GTGGACCTGATCATGGCGGGTCAAATCCACAAAG), ER{alpha}L379A (sense primer 5'-GATCAGGTCCACCTTGCAGAATGTGCCTGGCTAG and antisense 5'-CTAGCCAGGCACATTCTGCAAGGTGGACCTGATC), ER{alpha}G521A (sense primer 5'-GGCCATGAGTAACAAAGCAATGGAGCATCTGTACAGC and antisense 5'-GCTGTACAGATGCTCCATTGCTTTGTTACTCATGGCC), ER{alpha}H524A (sense primer 5'-GTAACAAAGGCATGGAGGCACTGTACAGCATGAAGTGC and antisense 5'-GCACTTCATGCTGTACAGTGCCTCCATGCCTTTGTTAC).

Cell Culture and Transient Transfections—Mammalian two-hybrid experiments were performed as described in Ref. 15. HuH7 (human liver) cells were maintained in Dulbecco's modified Eagle's medium high glucose (Invitrogen) supplemented with 10% fetal bovine serum and 2 mM L-glutamine. For transient transfection, cells were seeded into 24-well plates 24 h before transfection in phenol red-free medium supplemented with 10% Dextran Charcoal-stripped fetal bovine serum and 2 mM L-glutamine. Cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Corp.). After transfection, cells were treated with ligands for 16 h before assaying luciferase and beta-galactosidase activity.

Surface Plasmon Resonance (SPR)—Measurements were performed using a Biacore X instrument and streptavidin-coated sensor chips. All experiments were carried out at 25 °C in 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20. 50–120 response units of biotinylated {alpha}betaV peptide (biotin-SGSGPGSREWFKDML) was immobilized onto the chip surface. Qualitative binding experiments were performed by flowing liganded ER{alpha}LBDs (1 µM dimeric concentration) over the sensor chip for 2 min at 5 µl min-1. For the competition experiments, OHT-liganded ER{alpha}{Delta}H12 was preincubated with CoRNRER box peptide (DAFQLRQLILRGLQDD) at the desired molar ratio for 30 min prior to injection of the protein-peptide mixture over the {alpha}betaV sensor chip. Preincubation with an LXXLL-containing peptide (EKHKILHRLLQDS) was used as a control.

Crystallography—A truncated ER{alpha}LBD (ER{alpha}{Delta}H12; residues 305–533) mutant was used to facilitate crystallization. OHT- and RAL-liganded ER{alpha}{Delta}H12 LBD was prepared as previously described (15). Peptide complexes were assembled by incubating protein with a 1.5-fold molar excess of peptide followed by concentration using ultrafiltration. Additional peptide was added to obtain a final peptide:LBD molar ratio of 3:1. Initial screening of crystallization conditions was performed at 19 °C in a 300-nl, 96-well sitting drop format using a Mosquito® liquid-handling robot (TTP Labtech). A single crystal of the ER{alpha}{Delta}H12·RAL·CoRNR box complex was grown from a 300-nl drop containing an equal mixture of protein (7 mg/ml) and reservoir solution of 0.35 M (NH4)2SO4, 0.7 M Li2SO4, 0.07 M tri-sodium citrate, pH 5.6. Crystals of the ER{alpha}{Delta}H12·OHT·{alpha}betaV complex were grown in hanging drops comprising equal volumes of protein (10 mg/ml) and reservoir solution of 2.5% (v/v) polyethylene glycol 550 monomethylether, 2.5% (w/v) polyethylene glycol 20000, 0.06 M calcium acetate, 0.1 M Tris, pH 8.5.

Crystals were cryoprotected by passing through a mother liquor solution supplemented with 25–30% (v/v) ethylene glycol prior to vitrification in liquid nitrogen. X-ray diffraction data were recorded on a Quantum-4 CCD detector at 100 K at the European Synchrotron Radiation Facility (Grenoble, France) and were processed using the HKL suite of programs (17). The structures were solved by molecular replacement with AMoRe (18) using the coordinates of ER{alpha}{Delta}H12·OHT (15); Protein Data Bank code 2BJ4 [PDB] ) as a search model. The peptides were clearly visible in the initial sigmaA-weighted electron density maps. Model building was performed with QUANTA (Accelrys, San Diego, CA), and the complexes were refined with REF-MAC (19) using appropriate non-crystallographic symmetry and translation/libration/screw parameter restraints. Data collection and refinement statistics are given in Table 1.


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  		TABLE 1
Data collection and refinement statistics

 
Analysis—Coordinates were superposed in QUANTA, and secondary structure assignment was carried out using DSSP. The peptide binding interfaces were analyzed with the help of the protein interfaces PISA server at the European Bioinformatics Institute (www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Figs. 2 and 3, A–C, were generated using the program PyMol (Delano Scientific; pymol.sourceforge.net/).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Corepressor Motifs Bind to a Common Site in ER—Previous studies have reported the isolation and identification of a number of short peptides using phage display that specifically recognize OHT-bound ER (11–13). The OHT-specific {alpha}betaV peptide was isolated from a random phage display peptide library (12), whereas the CoRNRER box peptide was isolated from a focused library based on the corepressor consensus motif (13). Although both peptides were affinity-selected using OHT-bound ERs, they exhibit differing abilities to interact with full-length ERs in a mammalian two-hybrid assay (Fig. 1B). The {alpha}betaV motif displayed a robust interaction with full-length ER in the presence of OHT comparable with 17beta-estradiol (E2)-dependent LXXLL motif binding (Fig. 1B). In comparison, the CoRN-RER box peptide provoked a much weaker reporter response. Removal of helix H12 from full-length ER (ER{alpha}{Delta}H12) significantly enhanced binding of both peptides and had a dramatic effect on the CoRNRER motif interaction (Fig. 1C). Furthermore, real-time binding analysis between ER-LBD and an immobilized {alpha}betaV peptide using SPR demonstrated that, in the context of the LBD alone, no interaction occurs unless H12 is removed (Fig. 1D). Competition studies using SPR indicated that both peptides target similar binding sites on the LBD surface, as preincubation of the LBD with one peptide reduced the binding to a peptide sensor chip (Fig. 1E). Similar binding behavior was observed with ERbeta (data not shown).


Figure 1
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  		FIGURE 1.
Affinity-selected peptides bind to overlapping sites on ER LBD. A, chemical structures of SERM ligands used in this work. The antagonistic side chains of 4-hydroxy-tamoxifen (OHT) and raloxifene (RAL) are shaded in gray. B, interaction of Gal4-DNA binding-tagged peptides with VP16-tagged ER{alpha}. Data presented as % activity with the LXXLL peptide interaction with wild-type ER in the presence of 17beta-estradiol set to 100%. C, comparison of Gal4-DNA binding-tagged CoRNRER box peptide interaction with full-length and H12-truncated (ER{alpha}{Delta}H12)ER{alpha}. Data presented as % activity with the peptide interaction with wild-type ER in the presence of OHT set to 100%. D, interaction of {alpha}betaV peptide with ER LBD using SPR. Sensorgrams shown were obtained from injection of 1 µM wild-type or H12-truncated ({Delta}H12)ER{alpha}LBD, liganded to either 17beta-estradiol or OHT, over a surface immobilized with {alpha}betaV peptide. E, competitive binding studies using SPR. Sensorgrams obtained from injection of 1 µM ER{alpha}{Delta}H12·OHT over an {alpha}betaV surface. ER{alpha}{Delta}H12 was incubated with increasing molar ratios of CoRNR peptide prior to injection.

 
Previous studies by a number of groups have concluded that regions outside the LBD contribute to corepressor binding by NRs (10, 21–23). We also performed extensive domain deletion experiments as well as single site mutations to further investigate the architecture of the corepressor binding site (Fig. 5C and supplemental material). Domain mapping experiments suggest that the presence of ER's N-terminal AB domain is also required for interaction as constructs lacking this region exhibit no apparent binding to either CoRNRER or {alpha}betaV peptide in our M2H assay. As removal of H12 potentiates corepressor binding (Fig. 1, C–E), the role of the AB domain appears to be indirect, presumably through an effect on the positioning of H12 in the intact receptor that alters the accessibility of the AF2 binding site in the SERM-bound state. Interestingly, even though structural evidence does not exist, there are data indicating a functional communication between distant receptor N-terminal domains and the LBD (24).

Structure Determination—Initial co-crystallization trials with peptide and either OHT-liganded ER{alpha}-orERbetaLBD did not yield any crystals suitable for structural studies. Based on the SPR observations that minimal peptide binding to the isolated LBD occurs in the presence of H12, an H12-truncated ER{alpha}LBD (ER{alpha}{Delta}H12) mutant, in which 21 amino acids at the C terminus of the LBD encompassing the H11–12 loop and H12 were removed, was used to facilitate crystallization of the LBD·peptide complexes. The resulting structures of antagonist-bound ER{alpha}LBD in complex with either the CoRNRER box (DAFQLRQLILRGLQDD) or the OHT-specific {alpha}betaV (SPGSREWFKDMLS) peptides were solved by molecular replacement and refined to resolutions of 2.55 and 2.1 Å, respectively (Table 1).


Figure 2
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  		FIGURE 2.
Peptides bind along AF2 site. A, overall structure. ER{alpha}{Delta}H12 is shown schematically in gray. Binding modes of CoRNRER box (purple) and {alpha}betaV(green) peptides are highlighted. B, detailed view of the CoRNRER peptide binding site between H3 and H5. The peptide is colored cyan. For clarity, some side chains have been omitted. Peptide residues are labeled in black with one-letter amino acid codes. LBD residues that are buried on CoRNRER-box binding are colored green. Hydrogen bonds are drawn as dotted lines.

 


Figure 3
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  		FIGURE 3.
Structural mimicry of motif binding to AF2. A, the orientation of the CoRNRER box peptide (purple) is shown in comparison with silencing mediator of retinoid and thyroid receptors (SMRT) ID2 CoRNR box motif (green) from antagonist-bound PPAR{alpha} complex (Protein Data Bank code 1KKQ (10)). B, ER{alpha} H12 (green) in its antagonist conformation (Protein Data Bank code 3ERT (7)). C, {alpha}betaV peptide (green). D, structure-based sequence alignment of corepressor motifs and ER{alpha} H12. Boxed regions indicate key contact points with the AF2 binding groove. The lengths of the various helical elements are depicted above the alignment.

 
Structure of ER{alpha}{Delta}H12·RAL· CoRNRER Box Complex—The overall structure of the {Delta}H12 LBD and the conformation of raloxifene (Fig. 1A, RAL) is identical to that observed in the full-length ER{alpha}LBD·RAL complex (8). The CoRNRER box peptide is bound along the AF2 groove between H3 and H5 as an extended, 3.5-turn amphipathic {alpha}-helix (Fig. 2). The peptide, which is 20 Å in length, occupies the entire AF2 cleft with its N terminus extending into the position normally occupied by H12 in its agonist orientation. The canonical Leu/Ile residues of the CoRNRER motif (Leu5 (L+1), Ile9 (I+5), Leu13 (L+9) lie along one face of the helix and are buried at the interface between the LBD and peptide (Fig. 2B). Contacts with the LBD are almost exclusively non-polar in character. The N terminus of the peptide is packed against the antagonist side chain of RAL. L+1, I+5, and L+9 form a hydrophobic keel that anchors the peptide in the AF2 groove. L+1 contacts Leu-354, Trp-383, and the terminal piperidine ring of the side chain of RAL. The central I+5 makes contact with Ile-358, Val-376, and Leu-379. L+9 is bound in a shallow depression formed by the side chains of Ile-358, Phe-367, Leu-372, and Val-376. These van der Waals interactions are complemented by two polar interactions. Lys-362 is hydrogen-bonded to the main chain carbonyl group of Gly-12 and provides electrostatic stabilization to the C-terminal end of the CoRNRER box. In addition, Glu-380 participates in a salt bridge with the side chain of Arg-6 (Fig. 2B).

Comparison of the ER CoRNRER complex with the structure of PPAR{alpha}LBD bound to antagonist and the ID2 CoRNR box motif of SMRT (10) reveals that the general principles of corepressor motif binding to the AF2 region of NRs are conserved. Nonetheless, several notable differences are apparent because of the differing surface topology of the AF2 regions of these two NR LBDs (Fig. 3A). The SMRT ID2 helix is shorter than the ER motif and is severely distorted at its N terminus so as to maintain favorable packing contacts with the AF2 cleft of peroxisome proliferators activated receptor (PPAR). In particular, differences in the amino acid composition of the H5/6 junction dictate that the Leu+1 (Leu-685) residue of the SMRT motif binds closer to the LBD surface. In addition, whereas the bulky side chain of RAL protrudes from the ligand binding cavity and interacts with L+1 of the CoRNRER motif, the bound antagonist in the PPAR-SMRT structure contributes very little to the immediate CoRNR binding surface.

Role of H12 in CoRNR Box Binding—The inhibitory properties of H12 on CoRNR box binding to ER (Fig. 1C) are readily apparent when one compares the interaction modes of these two elements. Both peptides interact with the LBD in a fashion similar to that observed for H12 in complexes of the intact ER{alpha}LBD bound to SERM AF2 antagonists such as RAL and OHT (Fig. 3B). A structure-based sequence alignment clearly highlights the similarity between the affinity-selected CoRNR box motif and the ER{alpha} sequence in the vicinity of H12 (Fig. 3D). The CoRNRER box motif's Leu+1, Ile+5, and Leu+9 perfectly mimic the equivalent interactions made by the Leu-536, Leu-540, and Leu-544 of H12. This observation may also explain the apparent inability of ER to bind CoRNR box sequences found in bona fide NR corepressors such as N-CoR and SMRT (13, 25). In effect, ER possesses its own, highly effective CoRNR box surrogate within H12 that preferentially occupies the AF2 site in the presence of SERMs and passive antagonists. Consequently, H12 would need to be displaced from AF2 before any CoRNR box-mediated, corepressor binding could occur. Nonetheless, corepressors are understood to play a significant role in the biological effects of ER antagonists. SERM-bound ER{alpha} has been shown to be associated with N-CoR/SMRT in vivo (5, 26, 27); however, based on our study it seems highly unlikely that such associations are directly mediated through the AF2 region. Importantly, both N-CoR and SMRT have been isolated as part of multiprotein complexes (28, 29) and are more likely to be recruited to ER{alpha} via indirect mechanisms that require additional factors.

H12 Length as a Predictor of Corepressor Binding—Examination of the sequences in the vicinity of H12 suggests that this mechanism to resist corepressor binding may be quite common within the NR superfamily (Fig. 4). NRs that exhibit poor corepressor binding, such as RXR, have a H12 sequence that resembles the CoRNR consensus motif and are able to adopt a relatively long amphipathic helix that, like ER, would be able to occlude the entire AF2 binding site (CoRNR-class in Fig. 4). In the case of DAX-1 and SHP there is strong support for a CoRNR box corepressor-independent repression (reviewed in Refs. 30, 31). In contrast, NRs that exhibit good corepressor binding, such as thyroid receptor, peroxisome proliferators receptor, liver x receptor, retinoid acid receptor (10, 22, 32–34) exhibit much less similarity to the CoRNR box sequence. Critically, these NRs all possess a H12 sequence that is incompatible with the formation of a long AF2-blocking helix due to the presence of a proline residue that restricts the length of H12 (CoRNR+ class). The resultant shorter H12 would constitute no barrier to corepressor binding as CoRNR box-containing corepressors are more likely to be able to displace H12 and bind along the AF2 groove of these NRs. Structural data to support such a mechanism to resist corepressor binding are limited as there are relatively few examples of crystal structures of antagonist-bound NRs, other than with ER, in which H12 is observed to occupy the AF2 cleft. Nonetheless, in the structure of the retinoid x receptor/retinoid acid receptor heterodimer (35), H12 of both partners lies in the antagonist position but the helix of retinoid x receptor is considerably longer and buries 20% more accessible surface area than that of retinoid acid receptor. Consequently, corepressors are more likely to be able to displace retinoid acid receptor H12 and preferentially bind along its AF2 groove within this heterodimer. Notably, androgen receptor (AR), glucocorticoid receptor (GR), mineral-corticoid receptor (MR), and progesterone receptor (PR) contain an intermediate H12 length that does not harbor a complete CoRNR box-like sequence and would allow corepressor binding. In light of recent reports describing interactions ("direct" or "indirect") between NCoR/SMRT and these steroid receptors, it would be interesting to reinvestigate the possibility of direct recruitment to LBD (23, 36–39). The role of sequences C-terminal to H12 is unknown, but long extensions may influence both H12 stability and coregulator access.


Figure 4
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  		FIGURE 4.
Helix H12 sequence as a predictor of CoR binding to NRs. Comparison of the amino acid sequences of a range of NRs in the vicinity of H12. Sequences are shown beginning at the proline residue (red) that typically defines the N-terminal extent of H12. The core region of H12 is highlighted. The corepressor CoRNR consensus motif is boxed. NRs that are known to be poor binders of corepressors show a strong similarity between the sequence of H12 and the CoRNR motif. Sequences are subdivided into two classes based on H12 length (CoRNR- and CoRNR+). Sequences indicated by asterisks have a C-terminal extension of either 20 amino acids (*) or more than 30 amino acids (**).

 
Structure of ER{alpha}{Delta}H12·OHT·{alpha}betaV Peptide Complex—Despite bearing little sequence homology to the CoRNR box consensus sequence, the OHT-specific {alpha}betaV motif acts as an effective structural mimic of a corepressor motif and binds along ER's AF2 groove in a similar fashion (Fig. 2A). The {alpha}betaV peptide adopts a compact two-turn helix with a non-helical, N-terminal extension that sits atop the dimethylamino headgroup of the basic side chain of OHT (Figs. 3C and 5A). Whereas the CoRNRER box peptide uses three hydrophobic Leu/Ile residues to bind to AF2, the {alpha}betaV motif utilizes only two. Phe-8 and Leu-12, which are characteristic of the {alpha}betaV class of motif (16), occupy analogous spatial positions to CoRNRER box I+5 and L+9 residues. Trp-7 packs against the face of Phe-8, clamping the phenylalanine into a deep pocket in the AF2 groove (Fig. 5A). This structural feature appears to be important for the binding of the motif and explains why this position is always occupied by an aromatic residue (16). The helical nature of the {alpha}betaV motif is further stabilized by the side chains of Pro-2, Trp-7, and Met-11, which interlock and form a hydrophobic stack that interacts with the H3 edge of the binding groove (Fig. 5A). Analysis of other SERM-specific peptides reveals that the majority contain a CoRNR-like consensus sequence enriched with bulky hydrophobic residues replacing the canonical Leu/Ile residues (14).


Figure 5
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  		FIGURE 5.
AF2 surface is required for CoR peptide binding. A, surface representation of the {alpha}betaV binding site. The molecular surface of ER{alpha}{Delta}H12 is shown. Peptide and LBD residues that are important for binding are highlighted. B, alanine scan of the {alpha}betaV motif. Each position of the {alpha}betaV peptide motif was replaced by alanine, and peptide binding was measured in the M2H system using full-length ER{alpha} in the presence of OHT. C, binding of ER{alpha} mutants to affinity-selected {alpha}betaV and CoRNRER peptides in mammalian cells. Data are presented as % activity where peptide interaction with OHT-liganded wild-type receptor is set to 100%.

 
The ability of {alpha}betaV to adopt a shorter, compact {alpha}-helical structure compared with the CoRNRER motif may account for its ability to efficiently compete with H12 for the AF2 site in full-length ER (Fig. 1B). Furthermore, the structure of the {alpha}betaV complex suggests that direct contact with the protruding side chain of OHT contributes to the observed ligand specificity of the peptide and provides a molecular basis for SERM-specific coregulator interactions. This ligand-dependent interaction is supported by mutagenesis data that show that either replacement of residues at the N terminus of {alpha}betaV that are in direct contact with OHT's terminal dimethylamino moiety, or mutation of the ER side chain involved in the positioning of the OHT side chain (Asp-351), abolish peptide interaction (Fig. 5B). The ER{alpha} mutation D351Y is the only naturally occurring receptor mutation found in a tumor cell line responsive to OHT-stimulated growth (40). This residue is suggested to be able to regulate both estrogenic and antiestrogenic properties of OHT-complexed ER (20, 41). Although the precise contribution of this residue to ER ligand pharmacology is not clear, it has been suggested that, by affecting the positioning of the basic side chain of OHT, this may provide an extra coactivator interaction site (41). An alternative explanation of the regulatory properties attributed to this residue is that its mutation leads to the disruption of a potential {alpha}betaV-like corepressor interaction surface.

To further investigate the specificity determinants of the novel {alpha}betaV motif, we performed alanine scanning mutagenesis and evaluated binding to full-length ER using a M2H interaction assay (Fig. 5B). Replacements of residues that lie on the hydrophobic face of the {alpha}betaV helix (Trp-7, Phe-8, Met-11, Leu-12) abolish binding. Similarly, mutations that disrupt the AF2 surface (L358R, L379R, L372R) or remove the "charge clamp" lysine (K362A) also abolished binding of both the CoRNRER and the {alpha}betaV peptides (Fig. 5C). Intriguingly, mutation of Asp-351 has a differential effect on peptide binding and significantly enhances the interaction of the CoRNRER box-containing motif while abolishing {alpha}/betaV binding (Fig. 5C).

Concluding Remarks—The two structures presented here demonstrate that the AF2 region of ER is, in principle, capable of interacting with coregulator proteins that recognize the SERM-bound conformational state of the receptor. However, the internal CoRNR box motif within ER{alpha}'s H12 serves as an effective "corepressor surrogate" and provides a considerable barrier to binding. In addition, this study shows that amino acid sequences, other than the classical CoRNR box, can bind to the corepressor site of ER. These observations raise the possibility that cofactors with the binding characteristics of {alpha}betaV may exist in vivo and contribute to the effects of SERMs as this interaction motif, in contrast to CoRNR box-containing factors, is readily recruited to full-length antagonist-bound ER.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2JFA and 2JF9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

2 Supported by grants from the Swedish Cancer Society and the European Network of Excellence CASCADE. Back

3 Supported by grants from the Swedish Research Council, the Swedish Cancer Society, and the European Network of Excellence CASCADE. Back

1 To whom correspondence may be addressed. Tel.: 47-8-6089155; Fax: 47-8-7745538; E-mail: nina.heldring{at}biosci.ki.se. 4 To whom correspondence may be addressed: Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Headington, Oxford OX3 7LD, UK. Tel.: 44-1865-227358; Fax: 44-1865-737231; E-mail: ashley.pike{at}sgc.ox.ac.uk.

5 The abbreviations used are: NR, nuclear receptor; AF, activation function; CoR, corepressor; ER, estrogen receptor; LBD, ligand-binding domain; OHT, 4-hydroxytamoxifen; RAL, raloxifene; SERM, selective estrogen receptor modulator; SPR, surface plasmon resonance; SMRT, silencing mediator of retinoid and thyroid receptors; PPAR, peroxisome proliferators activated receptor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Rosenfeld, M. G., Lunyak, V. V., and Glass, C. K. (2006) Genes Dev. 20, 1405-1428[Abstract/Free Full Text]
  2. Huang, E. Y., Zhang, J., Miska, E. A., Guenther, M. G., Kouzarides, T., and Lazar, M. A. (2000) Genes Dev. 14, 45-54[Abstract/Free Full Text]
  3. Dobrzycka, K. M., Townson, S. M., Jiang, S., and Oesterreich, S. (2003) Endocr.-Relat. Cancer 10, 517-536[CrossRef]
  4. Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (2002) Science 295, 2380-2381[Abstract/Free Full Text]
  5. Shang, Y., and Brown, M. (2002) Science 295, 2465-2468[Abstract/Free Full Text]
  6. Pike, A. C. W. (2006) Best Practice Res. Clin. Endocrinol. Metab. 20, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  7. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[CrossRef][Medline] [Order article via Infotrieve]
  8. Brzozowski, A. M., Pike, A. C. W., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J.-Å., and Carlquist, M. (1997) Nature 389, 753-758[CrossRef][Medline] [Order article via Infotrieve]
  9. Pike, A. C. W., Brzozowski, A. M., Walton, J., Hubbard, R. E., Thorsell, A. G., Li, Y. L., Gustafsson, J.-Å., and Carlquist, M. (2001) Structure 9, 145-153[Medline] [Order article via Infotrieve]
  10. Xu, H. E., Stanley, T. B., Montana, V. G., Lambert, M. H., Shearer, B. G., Cobb, J. E., McKee, D. D., Galardi, C. M., Plunket, K. D., Nolte, R. T., Parks, D. J., Moore, J. T., Kliewer, S. A., Willson, T. M., and Stimmel, J. B. (2002) Nature 415, 813-817[Medline] [Order article via Infotrieve]
  11. Heldring, N., Nilsson, M., Buehrer, B., Treuter, E., and Gustafsson, J. A. (2004) Mol. Cell. Biol. 24, 3445-3459[Abstract/Free Full Text]
  12. Paige, L. A., Christensen, D. J., Gron, H., Norris, J. D., Gottlin, E. B., Padilla, K. M., Chang, C. Y., Ballas, L. M., Hamilton, P. T., McDonnell, D. P., and Fowlkes, D. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3999-4004[Abstract/Free Full Text]
  13. Huang, H. J., Norris, J. D., and McDonnell, D. P. (2002) Mol. Endocrinol. 16, 1778-1792[Abstract/Free Full Text]
  14. Iannone, M. A., Simmons, C. A., Kadwell, S. H., Svoboda, D. L., Vanderwall, D. E., Deng, S. J., Consler, T. G., Shearin, J., Gray, J. G., and Pearce, K. H. (2004) Mol. Endocrinol. 18, 1064-1081[Abstract/Free Full Text]
  15. Kong, E. H., Heldring, N., Gustafsson, J. A., Treuter, E., Hubbard, R. E., and Pike, A. C. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 3593-3598[Abstract/Free Full Text]
  16. Norris, J. D., Paige, L. A., Christensen, D. J., Chang, C. Y., Huacani, M. R., Fan, D., Hamilton, P. T., Fowlkes, D. M., and McDonnell, D. P. (1999) Science 285, 744-746[Abstract/Free Full Text]
  17. Otwinowski, Z., and Minor, W. (1997) in Methods in Enzymology (Carter, C. W., Jr., and Sweet, R. M., eds) Vol. 276, pp. 307-326, Academic Press Inc., New York[CrossRef]
  18. Collaborative Computational Project Number 4 (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
  19. Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240-255[CrossRef][Medline] [Order article via Infotrieve]
  20. Yamamoto, Y., Wada, O., Suzawa, M., Yogiashi, Y., Yano, T., Kato, S., and Yanagisawa, J. (2001) J. Biol. Chem. 276, 42684-42691[Abstract/Free Full Text]
  21. Hu, X., and Lazar, M. A. (1999) Nature 402, 93-96[CrossRef][Medline] [Order article via Infotrieve]
  22. Marimuthu, A., Feng, W., Tagami, T., Nguyen, H., Jameson, J. L., Fletterick, R. J., Baxter, J. D., and West, B. L. (2002) Mol. Endocrinol. 16, 271-286[Abstract/Free Full Text]
  23. Wang, D., and Simons, S. S., Jr. (2005) Mol. Endocrinol. 19, 1483-1500[Abstract/Free Full Text]
  24. Metivier, R., Stark, A., Flouriot, G., Hubner, M. R., Brand, H., Penot, G., Manu, D., Denger, S., Reid, G., Kos, M., Russell, R. B., Kah, O., Pakdel, F., and Gannon, F. (2002) Mol. Cell 10, 1019-1032[CrossRef][Medline] [Order article via Infotrieve]
  25. Webb, P., Nguyen, P., and Kushner, P. J. (2003) J. Biol. Chem. 278, 6912-6920[Abstract/Free Full Text]
  26. Lavinsky, R. M., Jepsen, K., Heinzel, T., Torchia, J., Mullen, T. M., Schiff, R., Del-Rio, A. L., Ricote, M., Ngo, S., Gemsch, J., Hilsenbeck, S. G., Osborne, C. K., Glass, C. K., Rosenfeld, M. G., and Rose, D. W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2920-2925[Abstract/Free Full Text]
  27. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843-852[CrossRef][Medline] [Order article via Infotrieve]
  28. Yoon, H. G., Chan, D. W., Huang, Z. Q., Li, J., Fondell, J. D., Qin, J., and Wong, J. (2003) EMBO J. 22, 1336-1346[CrossRef][Medline] [Order article via Infotrieve]
  29. Zhang, J., Kalkum, M., Chait, B. T., and Roeder, R. G. (2002) Mol. Cell 9, 611-623[CrossRef][Medline] [Order article via Infotrieve]
  30. Bavner, A., Sanyal, S., Gustafsson, J. A., and Treuter, E. (2005) Trends Endocrinol. Metab. 16, 478-488[CrossRef][Medline] [Order article via Infotrieve]
  31. Lalli, E., and Sassone-Corsi, P. (2003) Mol. Endocrinol. 17, 1445-1453[Abstract/Free Full Text]
  32. Chen, J. D., and Evans, R. M. (1995) Nature 377, 454-457[CrossRef][Medline] [Order article via Infotrieve]
  33. Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., and Glass, C. K. (1995) Nature 377, 397-404[CrossRef][Medline] [Order article via Infotrieve]
  34. Wagner, B. L., Valledor, A. F., Shao, G., Daige, C. L., Bischoff, E. D., Petrowski, M., Jepsen, K., Baek, S. H., Heyman, R. A., Rosenfeld, M. G., Schulman, I. G., and Glass, C. K. (2003) Mol. Cell. Biol. 23, 5780-5789[Abstract/Free Full Text]
  35. Bourguet, W., Vivat, V., Wurtz, J. M., Chambon, P., Gronemeyer, H., and Moras, D. (2000) Mol. Cell 5, 289-298[CrossRef][Medline] [Order article via Infotrieve]
  36. Dotzlaw, H., Papaioannou, M., Moehren, U., Claessens, F., and Baniahmad, A. (2003) Mol. Cell. Endocrinol. 213, 79-85[CrossRef][Medline] [Order article via Infotrieve]
  37. Hodgson, M. C., Astapova, I., Cheng, S., Lee, L. J., Verhoeven, M. C., Choi, E., Balk, S. P., and Hollenberg, A. N. (2005) J. Biol. Chem. 280, 6511-6519[Abstract/Free Full Text]
  38. Wu, Y., Kawate, H., Ohnaka, K., Nawata, H., and Takayanagi, R. (2006) Mol. Cell. Biol. 26, 6633-6655[Abstract/Free Full Text]
  39. Yoon, H. G., and Wong, J. (2006) Mol. Endocrinol. 20, 1048-1060[Abstract/Free Full Text]
  40. Levenson, A. S., Catherino, W. H., and Jordan, V. C. (1997) J. Steroid Biochem. Mol. Biol. 60, 261-268[CrossRef][Medline] [Order article via Infotrieve]
  41. Levenson, A. S., MacGregor Schafer, J. I., Bentrem, D. J., Pease, K. M., and Jordan, V. C. (2001) J. Steroid Biochem. Mol. Biol. 76, 61-70[CrossRef][Medline] [Order article via Infotrieve]

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