Crystal structure of a mutant hERalpha ligand-binding domain reveals key structural features for the mechanism of partial agonism.

The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.

Steroid hormones regulate the transcription of target genes in the cell by binding to transcription regulators that belong to the superfamily of nuclear receptors. All members of this family display a modular structure composed of six domains (A-F). The E region constitutes the ligand-binding domain (LBD) 1 containing a ligand-dependant transactivation function (AF-2) (1,2). The transcriptional activity of nuclear receptors is mediated by interactions with the transcriptional machinery through various corepressors and coactivators (3). Their ability to modulate gene expression in a ligand-regulated manner is based on the position of helix H12 carrying the AF2-AD transactivation function (4). Several positions of H12 have been observed (5). In the absence of ligand, H12 has been shown to be exposed to solvent (6). Ligand binding triggers a conformational change that results in the repositioning of H12 on the core of the LBD, closing the ligand binding pocket like a lid (7). This is referred to as the mouse trap mechanism (8). In agonistbound LBDs a surface suitable for coactivator binding is then created (9 -12). In most antagonist-bound complexes (11,12), H12 has been observed positioned in a structurally conserved cleft where the LXXLL motif of the coactivator molecule binds. These observations suggest a mechanism for antagonism where H12 and the coactivator compete for a common binding site. Note that the agonist position of H12 is unique, whereas its position in antagonist-bound complexes is not. Therefore knowledge of the features responsible for inducing and stabilizing a given conformation is a key step in understanding the initial events of nuclear receptor transactivation.
Several crystal structures of both ER isotypes (ER␣ and ER␤) have been solved in complex with natural and synthetic ligands (12)(13)(14)(15)(16). The natural ligand 17␤-estradiol acts as a pure agonist on both isotypes. Others typified by EM-800 and ICI164,384 are described as pure antagonists (17). A third category of ligands displaying cell-type and promoter dependence in ER regulation are referred to as selective ER modulators (SERMs) (18). SERMs such as raloxifen and 4-hydroxytamoxifen efficiently antagonize the AF2, but not the AF1 function, and act as a pure antagonist (19) in ER␤, which seems to lack a functional AF1 domain (20). The features responsible for inducing a given conformation and stabilizing it are crucial to the definition of the optimal stereochemical and biophysical specificity of a ligand. Here we present the comparison of the wild type hER␣ LBD crystal structure (16) with that of a mutant protein complexed with estradiol, where three cysteine residues were mutated in serine. The mutant protein binds estradiol with wild type affinity but has limited transcriptional capacity. In the structure of the Cys 3 Ser triple mutant hER␣ LBD, we observed an antagonist conformation despite the presence of a tightly bound estradiol in the ligand-binding cavity. This antagonist conformation, together with the transcriptional activity of the single, double, and triple cysteine to serine mutant receptors, supports the view of the agonist-antagonist equilibrium of H12 and gives some insight into the molecular mechanism for the conformational switch that drives the receptor in an agonist or antagonist conformation.

EXPERIMENTAL PROCEDURES
Protein Production, Purification, and Crystallization-The Cys 3 Ser triple mutant hER␣ LBD (Lys 302 3 Pro 552 ), in fusion with six histidine residues is produced using the pET15b/Escherichia coli BL21(DE3) expression system and purified by a zinc affinity column, ion exchange, and gel filtration. The purification procedure is similar to that of the wild type ER LBD (16). Crystals were obtained by vapor diffusion at 4 and 17°C using hanging drops made by mixing 1 l of protein solution (2.5 mg/ml) with 1 l of reservoir solution (12% poly-* This work was supported by funds from Schering AG and CNRS and by grants from the Ministère de la Recherche et de l'Enseignement Supérieur and the Association pour la Recherche sur le Cancer. 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. The ethylene glycol 8000, 0.4 M NaCl, 100 mM imidazole, pH ϭ 6.9). Prior to data collection, crystals were flash-cooled in liquid ethane after a fast soaking in a cryoprotectant buffer (20% glycerol, 15% polyethylene glycol 8000, 0.4 M NaCl, 100 mM imidazole, pH ϭ 6.9). Crystals belong to the space group P6 5 22 with cell parameters a ϭ b ϭ 58.6 Å, c ϭ 276.02 Å, ␣ ϭ ␤ ϭ 90°, ␥ ϭ 120°(one monomer in the asymmetric unit, 45% solvent).
Structure Determination-X-ray data were collected at 120 K in a nitrogen gas stream using synchrotron radiation (European Synchrotron Radiation Facility Laboratoire pour l'Utilisation du Rayonnement Eléctromagnétique). The diffracted intensities were processed using the programs DENZO and SCALEPACK (21). Experimental phases were obtained using gold and platinum derivatives. The multiple isomorphous replacement analysis was performed using CCP4 (22) and SHARP (23) packages. It enabled the construction of the complete model. An initial map was calculated to 3-Å resolution using multiple isomorphous replacement phases and solvent flattening using SOLO-MON (22,23). Refinement was performed with CNS (24) using bulk solvent corrections. All data between 15-and 2.2-Å resolution were included with no sigma cutoffs ( Table I).
The dimer interface was calculated with the Grasp package (25). The buried interface, calculated with non-hydrogen atoms only, was obtained with the excluded area method, which calculates the accessible surface regions of protomer A buried by protomer B.
ER Expression Vectors and Reporter Plasmids-pSG5-HEGO (26), pG4M polyII-ER(DEF) (27) for full-length ER and GAL-ER eucaryotic expression, respectively, are used in transactivation assays. Vit-tk-CAT (28) for full-length ER transcriptional activity and 17m-tk-CAT (29) for GAL-ER activity are used as reporter plasmids. CMV-␤Gal served as an internal control to normalize for transfection efficiency. Bluescript KS ϩ plasmid was used as carrier DNA. The procaryotic expression system pET15b-LBD/BL21(DE3) (Novagen) was used for estradiol binding ability and structural studies of the LBD.
Mutagenesis and Cloning-The mutations were generated in different constructs to allow structural and functional studies. Cysteine to serine mutations at positions 381, 417, and 530 were introduced in the LBD cloned in NdeI-BamHI sites of pET15b by PCR-assisted mutagenesis, using Deep Vent DNA polymerase (Biolabs) and the appropriate oligonucleotides. Triple mutant Cys 3 Ser His-tagged LBD was used in the structural studies and ligand binding assays. For transactivation assays the single, double, or triple mutants, in all possible combinations, were brought into the full-length receptor (pSG5-ER26) by digesting the LBD with the restriction enzymes HindIII-BglII. This fragment of 252 base pairs sharing the mutation C381S and/or C417S was inserted in the HindIII-BglII sites of pSG5-ER. The presence of another BglII cleavage site in pSG5 causes the loss of a fragment of 547 base pairs. This BglII-BglII fragment with or without the C530S mutation was reinserted in the vector. The BglII-BglII C530S fragment was obtained by PCR-assisted site-directed mutagenesis. To remove the AF1 contribution, the triple mutant was also brought into the XhoI-BamHI sites of the pG4M vector by PCR cloning using the pSG5-ER triple mutant as template, leading to the eucaryotic expression of GAL-ER(DEF).
Based on structural observations, another triple mutant was designed (E339A,E419A,K531A). These mutations were successively generated by PCR mutagenesis in the DEF region (282-595) subcloned in the XhoI-BamHI sites of pG4M. These mutations were also brought in the LBD subcloned in the NdeI-BamHI sites of pET 15b. All constructs were verified by automated DNA sequencing.
Cell Culture and Transfection-COS1 cells, an estrogen receptordeficient cell line, were transferred in phenol red-free Dulbecco's modified Eagle's medium supplemented with 5% charcoal-dextran-treated fetal calf serum and antibiotics (40 g/ml gentamicin, 0.1 mg/ml streptomycin, 500 units/ml specillin). Cells were plated in six-well dishes (Costar) at a density of ϳ5 ϫ 10 5 cells/well in a humidified 5% CO 2 atmosphere at 37°C. The cells were transfected 5-6 h later by the calcium phosphate coprecipitation technique with 0.2 g of wild type or mutant receptor plasmid, 2 g of reporter plasmid, 0.5 g of CMV-␤Gal (internal control plasmid), and 7.3 g of Bluescript KS ϩ carrier DNA. These plasmids were mixed in 420 l of 10 mM Tris-HCl, pH 8, 0.05 mM EDTA. 60 l of 2 M CaCl 2 was added by dripping. The mixture was dripped in 2ϫ 480 l of HBS (280 mM NaCl, 50 mM HEPES, 1.5 mM Na 2 HPO 4 ⅐12H 2 O, pH 7.12) and left 30 min at room temperature before being dispersed on the cells. 300 l were taken per well. The precipitate remained in contact with the cells for 15 h. After this exposure, the cells were washed with phenol red-free Dulbecco's modified Eagle's medium and antibiotics. Cells were then incubated in culture medium containing the indicated concentrations of estradiol for 24 h. Lysis was achieved in 300 l/well in 10 mM MOPS, 10 mM NaCl, 1 mM EGTA, 1% Triton X-100, pH 6.5, for 30 min at room temperature. The cellular lysates were centrifuged for 10 min at 16,000 ϫ g.
CAT Assays-The CAT was quantified by enzyme-linked immunosorbent assay (Roche Molecular Biochemicals) according to the manufacturer's recommendations. The amount of CAT was standardized for transfection efficiency with the ␤-galactosidase activity in each lysate. The basal level was defined as the CAT activity in cells transfected with the reporter plasmid in the absence of receptor plasmid. Each experiment was performed at least three times in duplicate. Results are expressed in relative CAT activity in percent of maximal wild type receptor activity (Fig. 3).
Estradiol Binding Ability of the LBD-We have used the pET15b-LBD/BL21(DE3) system to produce hER␣ LBD for ligand binding assays. E. coli BL21(DE3) expressing native or mutant LBD were lysed by sonification in the binding buffer (1 M SB201, 50 mM NaCl, 50 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol). After centrifugation at 14,000 ϫ g during 1 h at 4°C, the soluble fraction (crude extract) was used for receptor quantification and dissociation constant (K d ) determination. The total amount of protein in the crude extract was quantified TABLE I Data processing, phase determination and refinement statistics of Cys 3 Ser triple mutant structure (P6 5  . This mixture was left on ice for 5 min and centrifuged at 12,000 ϫ g for 5 min. The supernatant was removed for scintillation counting (30). Specific binding was plotted against the volume of crude extract for receptor quantification. For the K d determination the crude extract was incubated with increasing concentrations (from 10 Ϫ10 to 10 Ϫ7 M) of radiolabeled estradiol at 4°C overnight. Each measure was done in triplicate for Scatchard analysis. The variation of B/F as a function of B was analyzed as described previously (31).

RESULTS
The Molecular Structure-The structure of the triple mutant ER␣ LBD (Fig. 1b) exhibits the predominantly ␣-helical fold observed for all nuclear receptors. The superposition over the wild type structure in complex with estradiol (16) (Fig. 1a) leads to an r.m.s. deviation of 0.54 Å over 211 C␣ atoms (H1, H3-H8, H9-H11). The most striking conformational difference between these two structures is the different positioning of helix H12 and the concomitant shortening of helices H3 and H11 (Fig. 1, a and b). In the mutant, the activation helix is in the antagonist position as observed in the raloxifen and tamoxifen complexes (12,13). Both antagonist structures superimpose very well to that of the mutant LBD. The r.m.s. deviation is 0.5 Å over 213 C␣ atoms (H1, H3-H8, H9-H11, H12) and 0.5 Å over 238 C␣ atoms (H1-H6, H7-H11, H12) for the raloxifen and tamoxifen complexes, respectively (Fig. 1c). The loop L8 -9 is not seen in raloxifen, and the structure of the loop L1-3 is closer to the triple mutant in the complex with tamoxifen than that with raloxifen. Wild type and mutant homodimers can be superimposed with a r.m.s. deviation value of 0.64 Å over 418 C␣ atoms. The helices H9 and H10 form the core of the interface and contribute to more than 70% of it. Despite this good match, the contributions of the secondary structure elements to the interface, spanning the helices H7 to H11, differ among the two forms. Due to the antagonist conformation of the mutant structure, helices H7, H9, and H10 exhibit a smaller contact surface area (1475 Å 2 ) compared with the wild type (1686 Å 2 ).
The Ligand Binding Pocket-The overall structure of the pocket is similar in the wild type and the Cys 3 Ser triple mutant. All side chains of the hydrophobic residues lining the pocket are at the same position. This explains the fact that the dissociation constants at equilibrium (K d ) between the wild type and the Cys 3 Ser triple mutant for estradiol are very close (Table II and Fig. 2). The main differences are found on the 17-OH and 3-OH side of estradiol (Fig. 3). Due to the antagonist position of H12, the cavity in the triple mutant is not sealed as in the wild type structure. On the O17 side (D-ring side) of estradiol, the cavity reaches the surface of the protein and results in a much larger volume than the wild type ligand binding pocket. This channel is partially filled with water molecules, forming numerous hydrogen bonds with the protein. On the 3-OH side (A-ring side) an open narrow tunnel filled with water molecules is present in the mutant. In the wild type, this tunnel is almost closed and only the water molecule interacting with the 3-OH of estradiol is present. In the Cys 3 Ser triple mutant the estradiol A-ring superimposes perfectly with its equivalent group in the wild type complex, whereas the D-ring is slightly shifted, as shown by the displacement of the C17, which moves 0.5 Å closer to helices H3 and H12 (Fig. 3).
The superposition of the Cys 3 Ser triple mutant with the hER␣-raloxifen complex reveals that Asp 351 , which anchors the ammonium moiety of raloxifen, adopts the same conformation in both structures. All the helices, including H12, match perfectly (r.m.s. deviation: 0.5 Å), the protruding chain of raloxifen fitting perfectly in the water channel observed in the mutant structure. Furthermore, in this structure, electron density could be observed for the loop 11-12, a region that was not seen in the ER LBD/raloxifen structure. This loop includes the C terminus of the shortened helix H11, in particular Lys 529 , which points toward the ligand binding pocket channel.
ER pure antagonists exhibit acidic moieties in their protruding chain and are thus unlikely to interact with Asp 351 as do raloxifen and tamoxifen. The present structure suggests Lys 529 as a potential hydrogen bond partner for pure antagonist ligands bearing sulfinyl-like (ICI182780) (32) or sulfonyl-like (RU58668) (33) groups in their protruding chain. Such a contact would cross the AF2 AD groove and hamper the agonist positioning of H12.
Cumulative Effect of Cys 3 Ser Mutations on ER Transactivation Potency-Interestingly each single Cys 3 Ser mutation FIG. 1. a, schematic drawing of a hER␣ wild type LBD with the bound estradiol. The cysteine residues are depicted as yellow spheres. The AF2-AD containing helix H12 (red) is in the agonist position. b, the C381S,C417S,C530S hER␣ triple mutant LBD complexed to estradiol. H12 (red) is in the antagonist position. The now serine residues are depicted as pink spheres. c, superposition of wild type, Cys 3 Ser triple mutant and the complex with tamoxifen (12). Regions that superimpose perfectly are represented in gray, whereas regions that differ among the structures are colored in yellow for wild type LBD, pink for Cys 3 Ser triple mutant, and cyan for the tamoxifen complex.
(C381S,C417S; C530S) contributes equally to the observed transactivation reduction for the triple mutant receptor (Fig. 4 and Table II). Each single Cys 3 Ser mutation decreases the full-length receptor's activity by about 20%, whereas double mutations reduce CAT activity by ϳ40%, and the triple mutant exhibits a 56% decrease. Maximal wild type activity could not be restored even in the presence of saturating estradiol concentrations. Moreover each time a cysteine is mutated to serine the ligand dose-response curve of the mutant receptor is slightly shifted to the right, leading to a 6-fold shift in the ligandefficient concentration to trigger half-maximal activity (EC 50 ϭ 4.0 Ϯ 1.5 nM) for the triple mutant, compared with the wild type receptor (EC 50 ϭ 0.7 Ϯ 0.1 nM, Table II). To investigate the contribution of the AF1 on transcriptional activity, we used the chimeric receptors (GAL-ER) wild type and triple mutant on which the contribution of the ligand-independent transactivation function AF1 is removed. The Cys 3 Ser triple mutant displays 48% activity compared with the wild type GAL-ER, which is very close to the value observed for the full-length triple mutant receptor (44%, Fig. 2b). These data showed that the residual transactivation activity in the triple mutant is not due to AF1. Antagonist competition assays with raloxifen revealed that this SERM represses more efficiently the estradiolstimulated CAT activity of the Cys 3 Ser triple mutant GAL-ER than that of the wild type (Fig. 2c). These data suggest that the activation helix can be more easily displaced from its optimal position in the triple mutant context than in the wild type.
Effect of C381S Mutation on H12-Core Interface-The cysteine mutation at position 381 induces a destabilization of the agonist position of H12, which is most likely due to a solvating effect. This residue is located in helix H4, and its side chain is directed toward the solvent and is located in the agonist binding groove of H12. This residue is accessible in the mutant structure where H12 is in the antagonist position. In the wild type structure the cysteine residue is precluded from the solvent by helix H12. In the present structure this residue, which is now serine, is still solvent-accessible and is involved in a water-mediated hydrogen bond network lining the helix H12 agonist binding groove. In the mutant receptor, a positioning of H12 in the agonist groove is possible but would require the desolvation of the serine residue, a process more energetically costly for a serine than for a cysteine.
Effect of C530S Mutation on Helix H11-The C530S mutation disrupts the hydrophobic contact between Cys 530 and Tyr 526 (Fig. 5b) and contributes to the shortening by one turn of helix H11 at its C terminus end, compared with the wild type structure. A serine residue exhibits different solvating properties and favors a coil structure with a surface-exposed side chain, as observed in the Cys 3 Ser triple mutant. The shortening of H11 and the subsequent lengthening of loop 11-12 allow H12 to reach the coactivator binding groove, as observed in the tamoxifen-ER complex.
Effect of C417S Mutation on Helix H3-In the wild type structure, Cys 417 is located in the rather flexible loop 6 -7. Its side chain is involved in numerous hydrophobic contacts with neighboring residues (it forms van der Waals contacts with Phe 337 inside an hydrophobic core composed of the N-terminal parts of H3 (Phe 337 , Leu 345 ) and of the ␤-sheet (Leu 410 , Leu 408 ; Fig. 5a). The substitution by a serine residue, by disrupting these hydrophobic contacts, is likely to be the triggering factor that induces the shortening of helix H3 by one turn at its N terminus. The conformational reorganization includes the last 10 residues of loop 1-3. Interestingly, the tamoxifen complex is nearly identical to the triple mutant in this region, whereas the raloxifen-bound structure exhibits a wild type conformation without shortening. Note that the overall Cys 3 Ser triple mutant structure is closer to that of the tamoxifen-bound LBD than to the raloxifen one. Nevertheless some differences remain between the mutant and tamoxifen structures, especially in the loop 6 -7 region, which is shifted by more than 3.0 Å (Glu 419 and Gly 420 ) toward the core of the protein. This large movement in the antagonist structure is most likely induced by tamoxifen, whose aromatic ring is almost perpendicular to the estradiol D-ring and superimposes on the position 17. This movement of loop 6 -7, filling the cavity, encroaches on the N terminus of helix H3, which thus adopts a conformation similar to that of the mutant protein.
The Antagonist Conformation Disrupts a Hydrogen-bonding Network Observed in Agonist Complexes-The shortening of helices H3 and H11 affects the surrounding structure, in particular a hydrogen bond network present in the wild type involving the conserved Glu 419 on the loop H6-H7, Lys 531 on the end of H11, and Glu 339 at the N-terminal part of helix H3 (Fig. 5c). The disruption of these interactions is a direct consequence of both C417S and C530S mutations. The triple mutant E339A,E419A,K531A, which is unable to form the hydrogenbonding network, further underlines the importance of this network for H12 positioning. We have analyzed the transcriptional capacity of this Ala mutant and compared it to the Cys 3 Ser mutations using GAL-ER constructs (Fig. 2b). The Ala triple mutant displayed 62% activity compared with the wild type receptor, which is comparable with the value of 64% observed with the C417S,C530S double mutant. The similarity of effects suggests the occurrence of similar structural features for H12 positioning. DISCUSSION One generally accepted mechanism of antagonism is that steric hindrance inhibits the natural agonist conformation and favors an alternative position for H12 that then occupies the binding site of coactivators in the H3/H4 cleft. The present study allows us to dissociate the steric effect from the others. Indeed the crystal structure of the Cys 3 Ser triple mutant LBD bound to estradiol adopts a typical antagonist conformation, whereas estradiol binding and transactivation are not impaired. Each single Cys 3 Ser mutation lowers the transcriptional activity by ϳ20%, showing that the structurally unrelated regions that are perturbed are equally important for ER␣ to fully respond to estradiol stimulation. The combination of these mutations results in an additive effect in the reduction of transactivation, leading to a triple mutant that is only half as potent as the wild type receptor. This cumulative effect of the Cys 3 Ser mutations further confirms the lack of cooper- ativity of the observed conformational changes. The higher amount of estradiol needed to stimulate transcriptional activity, compared with the lower concentration of raloxifen required to repress it, suggests that Cys 3 Ser mutations favor the switching of helix H12 toward a nonproductive conforma-tion. These results favor a dynamic model where H12 occupies two more or less favorable states, with the mutations or the ligand affecting the equilibrium. This H12 flexibility is also illustrated by the ER␤ bound to genistein complex (15). Genistein distinguishes itself from SERMs by its smaller size, allowing its total burying in the ligand binding cavity. In the crystal structure, helix H12 adopts a position shifted by 25°from the antagonist conformation observed in the raloxifen complex. Taken together with the highly disordered helix H12 in the ER␤-raloxifen crystal structure and similar observations in other systems (RXR-RAR), it is clear that antagonist positions are not unique. The RXR␣ F318A LBD-oleic acid complex (34), with a typical antagonist conformation for H12, bears even more resemblance to our present situation in that the ligand also does not prevent the agonist position. In this case too the antagonist conformation is in apparent contradiction with the transcriptional activity of the receptor and its ability to recruit coactivators (34). Furthermore an agonist position with a weak partial agonist is observed in the PPAR␥ structure (35). All these cases can be explained by a "flip-flop" mechanism for H12 positioning, the equilibrium between the H12 agonist and antagonist positions in the coactivator binding groove would then depend on the cellular context (nature and concentration of cofactors) (5).
The comparison of the wild type and the Cys 3 Ser triple mutant structures reveals the molecular interactions that are essential for stabilizing the agonist conformer. If these are weakened or disrupted by altering the protein hydration and/or conformation, the equilibrium is shifted toward an alternative nonproductive conformation, explaining the partial decrease in transcriptional ability. When estradiol binds to ER, it interacts through the 17␤-hydroxyl group with His 524 , which in turn forms a hydrogen bond with the peptidic carbonyl group of Glu 419 in the loop 6 -7. This glutamic acid contacts Glu 339 from helix H3 and Lys 531 from helix H11, forming a hydrogen bond network that favors the helix H12 agonist position (Fig. 5c). The loop 1-3 accompanies the movement of helix H3, as shown in other nuclear receptors. The precise positioning of H3 is an important feature for the constitution of the ligand binding cavity.

CONCLUDING REMARKS
The destabilization of the H12-protein core interaction is at the heart of the mechanism of partial and pure antagonism. The dominant effect depends on the potency of the ligand to disrupt the active conformation or in other words to prevent the correct binding of coactivators. The present study sheds light on the molecular mechanism and the structural basis of partial agonism on the AF2 transactivation function. The position of H12 can be modulated by the cellular context of cofactors, their ability to displace the equilibrium and to stabilize one conformer.
For the glucocorticoid and estrogen receptor, cysteine modi-fication plays a role in gene regulation by the intracellular redox potential modification (36 -38). The importance of cysteine residues located in the activation domain has recently been stressed for the nuclear factor I/CCAAT transcription factor (NFI/CTF) (39). Intracellular thioredoxin or metal ion concentration, which have high affinity for sulfhydril groups, would act as the regulator of the transcription. Their effect on transactivation could be explained by the modification of cysteines, linking hormonal and redox signaling pathways. ER, like other steroid nuclear receptors, is unstable in the absence of ligand or protein cofactors like HSP90 (33). The fold stabilization of these proteins is part of the control of gene expression and is ligand-dependent (induced fit mechanism) and controlled by the cellular context (redox potential, nature of a ligand, presence of interacting molecules like coactivators or corepressors).
FIG. 5. a, effect of C417S mutation on H3. Superposition of wild type (yellow) and Cys 3 Ser triple mutant (gray) emphasizing the shortening of H3 by one turn and the significant conformational change of the loop 1-3 are shown. b, effect of C530S mutation on H11. Superposition of wild type (yellow) and Cys 3 Ser triple mutant (gray), showing the shortening of H11 on the mutant protein is shown. c, superposition of wild type (yellow) and triple mutant (gray) ER LBD structures near the mutated residues. The ligand is anchored by His 524 that interacts with the carboxyl group of Glu 419 , a residue from L5-6. This glutamate contacts both the N-terminal end of H3 (Glu 339 ) and the C-terminal end of H11 (Lys 531 ). The hydrogen bond network connecting the estradiol O17, His 524 and Glu 419 , Glu 339 , Lys 531 in the wild type structure is shown. The effect of the C417S and C530S mutations are to shorten by one turn the Nterminal end of H3 and the C-terminal end of H11, respectively. This leads to the disruption of the hydrogen bond network. To confirm the relevance of this network, Glu 339 , Glu 419 , and Lys 531 were mutated in alanines and compared with Cys 3 Ser mutant receptor in transactivation assays.