From the
Structure Biology, Karo Bio AB, Novum, SE-141 57 Huddinge, Sweden, the ¶Department of Structural Biology, Abbott Laboratories, Abbott Park, Illinois 60064-3500 and the ||Departments of Medical Nutrition and Biosciences, Karolinska Institute, Huddinge University Hospital, Novum, SE-141 57 Huddinge, Sweden
Received for publication, December 13, 2002 , and in revised form, April 3, 2003.
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ABSTRACT |
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INTRODUCTION |
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The four steroid receptors, GR, the progesterone (PR), androgen (AR), and mineralocorticoid receptors, are very closely related. They all bind to response elements with the same degenerate consensus sequence (11), and there is considerable overlap in ligand specificity and action (1214). Progesterone is a glucocorticoid antagonist, and many synthetic progestins are also androgens. Glucocorticoids, and particularly the endogenous hormone cortisol, bind with similar affinities to both GR and the mineralocorticoid receptor, although aldosterone is a poor GR agonist. Thus, detailed structural and functional data will be needed to understand the specific function of these four steroid receptors. Despite the problems purifying and crystallizing GR-LBD, there is a plethora of functional data obtained by analysis of mutant forms of the receptor. These data have been collated by Simons (15) and provide a rich insight into the function of the LBD compared with almost all other members of the nuclear receptor family. To further understand the function of key residues of the GR-LBD and its interaction with the ligand, we have previously carried out a systematic functional analysis of mutations of specific sites within the GR-LBD (1618). This abundance of functional data should prove invaluable for understanding the mechanism of steroid hormone binding and action when compared with a specific structure for the GR-LBD. In this study we describe the structure of GR-LBD, both in association with the antagonist RU-486 as well as in association with the agonist dexamethasone.
The antihormone RU-486 (mifepristone) is an effective antiprogestin and antiglucorticoid that has shown clinical efficacy in both functions (19). It is also a weak antiandrogen. The function of the antagonistic action of RU-486 has been shown to be an active process and not just the blocking of agonist binding. Following the binding of RU-486, GR binds more tightly to specific DNA sequences with a slower dissociation rate (20). The antagonist ZK98299 appears to induce a differential PR conformation that affects the interaction with DNA (21). Thus, there is an interdomain functional interaction that is dependent on the ligand bound. Further evidence of this has been found with regard to ligand-dependent phosphorylation of GR. Although both dexamethasone and RU-486 induce phosphorylation of Ser-203, dexamethasone but not RU-486 induces phosphorylation of Ser-211 (22). This differential phosphorylation pattern was related to the intracellular location of the subspecies of GR. Binding of RU-486 blocks the binding of coactivators at the AF-2 site while simultaneously actively recruiting the binding of corepressor NCoR or silent mediator of retinoic acid receptor and thyroid receptor (SMRT) (23). This function is again dependent on the N-terminal domain of GR, although the corepressor interaction site is complex and involves sequences within the ligand-binding domain as well. In various model systems, RU-486 can act as an agonist in the absence of corepressor, acting through the N-terminal AF-1 site (23). The agonist function of RU-486 can also be shown for specific glucocorticoid-induced phenotypes, such as the induction of p27Kip1, part of the cytostatic action of glucocorticoids in osteosarcoma cells (24). A similar active antagonistic function of RU-486 has been shown with PR (for review, see Ref. 25). Thus, a detailed analysis of the differences between the structures of GR bound to dexamethasone compared with RU-486 is of importance to understand how the ligand exerts different biological functions through one single receptor protein.
Throughout this report, helix nomenclature from the original estrogen receptor structures (ER; PDB 1ERE
[PDB]
) will be used (26).
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EXPERIMENTAL PROCEDURES |
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Two systems were used to create the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV); BacVector (Novagen) and Bac-To-Bac (Invitrogen). GR was expressed using baculovirus-infected insect cells. Spodoptera frugiperda (Sf9) cells (Invitrogen) were maintained as suspension cultures in shaking flasks and routinely passaged every third day. The serum-free medium Sf900II (Invitrogen) was used with the addition of gentamicin (15 µg ml1, Sigma-Aldrich). A 100-liter stirred incubator tank (Belach Bioteknik AB) was used for large-scale expression. The cells were infected with recombinant A. californica nuclear polyhedrosis virus (Invitrogen) containing the gene encoding human GR-LBD (residues 500 777 expressed). The cells were harvested after 48 h and pelleted by centrifugation in a swing-out centrifuge at 2000 rpm, 20 min, 4 °C. After centrifugation the cell pellet was frozen in liquid N2 and stored at 70 °C.
Depending on expression levels, frozen cells harvested from 550 liters of culture volume were disrupted by thawing in the extraction buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 10 mM monothioglycerol (MTG), 50 µM dexamethasone) with a magnetic stirrer at 4 °C. The supernatant was recovered after centrifugation and was bound to TALON chelating resin (Clontech). The resin was washed to remove unbound material according to the manufacturer's guidelines. The GR-LBD was then eluted with 10 mM Tris-HCl, pH 8.0, 10% glycerol, 2.5 mM MTG, 50 mM imidazole, and 50 µM dexamethasone. The His6 tag was removed from N-terminal constructs by thrombin cleavage (10 units/mg GR) overnight at 4 °C. The cleaved protein was loaded on a Resource 30 Q cation exchange column (Amersham Biosciences) and subsequently eluted with a linear KCl gradient. The main peak was collected at 100 125 mM KCl and concentrated using a Centriprep-30 (Millipore) to 6 40 mg/ml. Native-PAGE analysis showed 9598% pure GR.
For the GR-LBD RU-486 structures, the pure protein was dialyzed (Slide-A-Lyzer, Pierce) for 48 h at 4 °C against 2x 600 ml buffer containing 10 mM Tris-HCl, pH 8.5, 2.5 mM dithiothreitol, and 50 µM RU-486. GR-LBD was finally concentrated to 512 mg/ml in a Centriprep-30 (Millipore) prior to crystallization.
CrystallizationCrystals were obtained by the vapor diffusion method in 23 µl of hanging drops containing equal volumes of protein and crystallization buffer. The following crystallization conditions were used. GR1 10% polyethylene glycol 8000, 80 mM CaCl2,50mM Tris-HCl, pH 8.8, 4 °C. GR2 1.5 M 1,6-hexanediol, 50 mM sodium citrate, pH 5.6, 2mM dithiothreitol at 12 °C. GR3 15% polyethylene glycol 8000, 900 mM 1, 6-hexanediol, 600 mM NaSCN, 100 mM Tris-HCl, pH 8.2, 12 °C. GR4 8 15% polyethylene glycol 400, 150 mM MgCl2, 0.2% dioxane, 25 mM Tris-HCl, pH 8.5, 8 °C and with five times molar excess of coactivator peptide TIF2, NR-box3 (NH2-KENALLRYLLDK-COOH, ordered from ThermoHybaid, Germany).
Data Collection and Structure DeterminationFor GR1 and GR2, diffraction data were collected at the Advanced Photon Source at Argonne National Laboratory (beamline 17-ID/Industrial Macromolecular Crystallography Association-Collaborative Access Team) using an attached MAR-CCD or ADSC Q-210 CCD detector, respectively. GR3 and GR4 data were collected on the beamline ID14 4 at the European Synchrotron Radiation Facility, Grenoble, France, also using an ADSC Q-210 CCD detector. All data were collected at 100 K.
GR1 and GR2 data were integrated and scaled with the HKL2000-package (27), whereas for GR3 and GR4, Mosflm and Scala were used (28). The GR1 structure was solved by molecular replacement in CNX (29) using a GR homology model based on coordinates from a PR monomer (PDB 1A28 [PDB] ) (8). The subsequent structures were solved using the best available GR structure at the time.
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RESULTS AND DISCUSSION |
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The Antagonistic Form, GR3This DNA construct included the F602S mutant, which has been reported to stabilize GR for Escherichia coli expression (30). The construct produced more protein in the baculovirus expression system as well (4 8 mg/liter). The crystals grew as rods to a maximum dimension of 280 x 80 x 80 µm. One crystal was cryocooled using the well solution but with 20% polyethylene glycol 8000 and 15% ethylene glycol.
The Agonist Form, GR4 Hexagonal looking crystals grew to a maximal size of 100 x 50 x 50 µm following the addition of coactivator peptide TIF2 NR box 3 to purified concentrated GR-LBD expressed with dexamethasone. The crystal proved very difficult to cryocool, and the best results were obtained by rapidly passing the crystal through well solution containing 20% glycerol as a cryoprotectant.
Structure Determination and RefinementThe third crystal form, GR3, the best one to date, was solved in Molrep (28), using a monomeric model of GR without helix 12. One large peak appeared in the rotation function map. A systematic search in the translation function revealed the P22121 solution that was very apparent with an R-factor of 52% and a correlation coefficient of 34%. After transformation to the standard setting in 21212, refinement was started in CNX. The R-factor dropped during the initial round of rigid body, B-factor, and slow cool refinement to 39% without any manual model building. RU-486 ligands were included in the CNX refinement by building the necessary libraries using XPLO2D (31). During iterative refinement, the missing part of the structure was readily built with the Grab_build command in O version 8.04 (32). Four 1,6-hexanediol molecules could be identified from a 2 Fo Fc map and subsequently built in the electron density. The final structure was refined to 22.0% R-factor (26.2% Rfree) with good stereochemistry and has been validated in Procheck (28) and OOPS2 (33). In the Ramachandran plot, only 4 residues are outliers (1.8%) (34). Two of them, Glu-705 and Ser-708, are in a badly defined loop.
For the agonist structure GR4, a brute force systematic molecular replacement search to locate four molecules using Molrep was conducted in all possible indexed space groups with a monomeric model based on the GR3 structure and the last 40 residues of the PR structure starting from the middle of helix 11, where GR3 and PR begin to diverge. A convincing solution with three molecules in the asymmetric unit was found consisting of a dimer and a single monomer in the space group P31. The program could not find the last molecule. This solution had about 74% solvent content but refined well with a rapid decrease of the R-factors. After rigid body refinement of the three molecules, calculation of an electron density map of the complete asymmetric unit revealed unoccupied density from a fourth molecule. Using Molrep with input of fixed calculated structure factors from the three molecules, the fourth molecule could be found. Now the asymmetric unit consists of a pair of dimers. Refinement was initially carried out using Refmac (28) and halted at r of 31% and Rfree of 34%. Twinning analysis using Detwin (28) revealed a 710% twinning factor with the matrix (k,h,l). This agrees with the fact that the data scale reasonably well in the apparent space group P321. Detwinning the data and continued refinement with Refmac show only a modest improvement of the R-factors, (31%/33%). Moving the refinement of the original data to CNX (29) and using the least squares residual for hemihedral twinning as refinement target allowed an immediate decrease of both R-factors to 24 and 27%, respectively. Even more encouraging, the resulting maps after simulated annealing and positional and grouped B-factor refinement allowed four more residues to be placed at the N terminus in three of the four molecules in the asymmetric unit. The structure is of worse overall quality compared with GR3; there are missing residues, 705707, and a loop including residues 615 619 involved in crystal contact is badly defined in density. (Table I).
The GR-LBD structures consist of three layers of -helices arranged in an antiparallel sandwich fashion (Fig. 2d). The overall structures of both antagonistic and agonistic GR are similar to those of other nuclear hormone receptors, in particular to the progesterone receptor (PR, PDB 1A28
[PDB]
) (8). GR4 (dexamethasone) and PR (1A28
[PDB]
) can be superimposed using LSQMAN (35) with a root mean square deviation fit on C
atoms of 0.79 Å for residues GR531775 and PR686 931. With GR3 (RU), PR aligns with an r.m.s. of 0.98 Å for residues GR530 734 and PR686 889 (helix 12 not included). The aligned structures share a 56.2% sequence identity. In GR3, the dimethylaniline side chain of RU 486 physically prevents helix 12 from adopting the characteristic agonist position over the ligand-binding pocket as in the GR4 (dexamethasone) structure. Differences can be seen especially with regard to the antagonist-induced conformational change C-terminal of residue Asn-734. In the GR3 structure, clear electron density can be seen for most of the amino acids ranging from amino acids 530 776 of the full-length receptor sequence, as well as the RU-486 molecule (Fig. 1). There is one missing loop (amino acids 760 767) after helix 12 in GR3. Residues in the loop amino acids 704 708 show weak electron density in all structures.
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Models have been published of GR based on the ER and the PR agonist structures (18, 36), but to model an antagonist conformation from an agonist one is difficult because of the large conformational change of helix 12 that can be seen, for example, in the ER raloxifene structure (26). In that case, binding of an antagonist to the LBD results in displacement of helix 12 so that it prohibits binding of the coactivator.
The dimethylaniline side chain of RU-486 prohibits binding of helix 12 in the agonist position as it is seen in the GR4 structure. In GR3, helix 11 is preterminated at Asn-734, whereas in GR4 the helix 11 continues to Asp-742. Instead of a helix, the structure is stretched out and the internal Cys-736 is translated over 4 Å on C and the side chain swings out toward the surface, going from psi = 120 to 52 degrees, i.e. a 172 degree rotation. The Cys-736 is within Van der Waal contacts with the ligands in all other structures, including that of the progesterone receptor. In GR3, the carboxyl oxygen of Cys-736 is connected to the 17
-hydroxyl group of the RU-486 molecule (Fig. 1A). Interestingly, an intermolecular disulfide is formed between the two Cys-736 residues over the crystallographic 2-fold axis, thereby rigidifying the loop between helices 11 and 11a (Fig. 2a). Helix 12 enters the other LBD subunit of the LBD dimer and binds in a hydrophobic cavity situated between the agonist position of helix 12 and the coactivator pocket. The orientation of helix 12 in GR3 is opposite to that of the traditional agonistic position like that of helix 12 of GR4. Superimposing the GR3 and GR4 structures reveals that the GR3 helix 12 is bound perpendicular to the beginning of the helix 12 position of GR4. Only Phe-749 and Met-752 reach into the same hydrophobic pocket that is occupied by Ile-756. A similar arrangement has previously been observed in the case of hER
(37) where, interestingly, a cystine at the end of helix 11 was also formed. It is unclear whether this disulfide is formed only in the crystals or only at high protein concentration or whether it has any biological implications. Tanenbaum et al. (37) drew the conclusion that it is probably an artifact from crystallization.
Cys-736 may be involved in interactions with heat shock proteins during folding of GR. Mutational studies of this residue have shown that it can only be moderately changed and that its steric properties are most important. C736T increased the affinity of the synthetic ligand triamcinolone acetonide but resulted in lower affinity for cortisol. C736S decreased transcriptional activity, and the C736G mutant did not express although bulkier side chains produced inactive GR (16).
To our surprise, a long stretch of electron density was seen on the surface of GR3 LBD between helices 9 and 11. This stretch of 10 residues perfectly matches the position of the C terminus of GR in the agonist complexes of GR with dexamethasone (GR4) and PR with progesterone (PDB 1A28
[PDB]
), showing that this arrangement can also be seen in an antagonized structure. There is a 16 Å distance (C-C
) between the last visible helix 12 residue (Asn-759) and the first residue in the C-terminal part (Asn-769). This distance corresponds to approximately nine flexible residues, indicating that helix 12 enters the neighboring molecule and then folds back, returning with its C terminus to its parent molecule. This tail seems to be important in stabilizing the GR protein. Constructs with shorter C-termini showed no or very low expression levels. In the case of ER, structures with the corresponding residues after helix 12 (the F-domain) show no traces of these residues in the electron density maps.2
The RU-486 molecule is bound in the same general way as other steroid hormone molecules in nuclear receptors (Fig. 1A). The steroid core is mainly held in place by hydrophobic residues that outline the cavity. Two direct hydrogen bonds from the 3'-carbonyl oxygen in the A-ring of the steroid core to Gln-570 and Arg-611 and a water molecule orient the steroid. At the other side, the 17-hydroxyl group makes a hydrogen bond to Gln-642 and links a water molecule to the carbonyl of Cys-736. Tyr-735, which has been shown to be important for transactivation (36), is a semi-conserved residue (PR = Tyr, AR = Phe) that occupies a similar position in the PR and AR structures. When comparing antagonist and agonist structures, however, a completely different rotamer is observed for Tyr-735. In the agonist structure it is within van der Waals contact with the ligand and forms a 3.3 Å hydrogen bond to Gln-642 to help stabilize its position in the binding pocket. In the RU-486 structures, though, Tyr-735 turns away from the ligand and forms hydrogen bond interactions with Asp-641. Overall, our data clearly show that in the GR4 structure helix 12 adopts a position over the ligand-binding pocket, allowing the TIF2 NR box 3 peptide to bind in the co-activator cavity forming a transcriptionally active receptor. However, in GR1 and GR3 the dimethylanaline side chain of RU-486 physically prevents helix 12 from adopting the characteristic agonist position as in GR4. Instead, helix 12 is translocated and now covers the co-activator cavity, thus preventing the receptor from recruiting a co-activator.
Cys-638 is a surface-exposed residue that has been extensively studied with the aid of site-directed mutagenesis (e.g. see Refs. 3840). In our early work with GR we treated the protein with iodoacetic acid to carboxymethylate exposed cysteines in much the same manner as the previously successful work on the estrogen receptors (26, 41). GR never responded very well to this treatment, and the resulting protein was not homogeneous enough for crystallization. To overcome the problem, Cys-638, which probably was the main target for carboxymethylation in the form of GR, was mutated to an aspartic acid in all of our later constructs, resulting in increased homogeneity. A positive side effect of the reaction was that the isoelectric point of the protein became lower with increased solubility as a result.
Another mutation that was introduced during our work to increase the stability of GR was F602S. This is an internally buried phenylalanine, which stretches into a hydrophobic pocket. In PR, this residue is a Ser but most of the other residues lining this pocket are conserved between PR and GR; the published GR structure from Bledsoe et al. (10) also contains this F602S mutant. Our GR1 and GR2 structures have the wild type Phe at this position. Comparisons of GR2 with GR3 or GR4 show that the serine mutation initiates a series of side chain movements in the vicinity of residue 602. His-726, which is involved in the water-mediated hydrogen bond network, swings away and Ser-673 makes a water-mediated hydrogen bond to Ser-602. In the wild type GR, Tyr-598 closes in and forms stacked interaction with the aromatic ring of Phe-602 (Fig. 3).
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The GR3 crystallization conditions contain 1,6-hexanediol, and in the refined electron density map three hexanediol molecules could be placed. The inclusion of 1,6-hexanediol does not seem to change any main chain conformations as seen on a superposition of GR3 and GR4 (Fig. 4.)
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The agonist structure GR4 was solved in P31 in contrast to the P61 structure that was recently published (10). The lower symmetry imposes four molecules in the asymmetric unit (Fig.2b). From that we show two plausible dimers (Fig. 2, c and d). The dimer shown in Fig. 2d is the species Bledsoe et al. (10) postulate to be the GR dimer as deduced from mutational studies as well as buried surface area calculation. In our GR work the nature of the GR dimer varies from structure to structure. Savory et al. (42) have shown that GR dimerization in solution seems dependent upon the intact hinge region but independent of the agonist/antagonist state of the protein. In our hands, all recombinant GR-LBDs that have so far produced diffraction quality crystals lack parts of the hinge region. The longest protein contains about half the hinge region, but this entity is not visible in the electron density maps. In all four structures, the electron density starts at residue 530 or 531. Our findings suggest that the hinge region is important for stabilizing the protein during fermentation and purification because constructs with shorter N termini expressed poorly.
The ligand is well defined in the electron density map (Fig. 1B). Superposition of the ligand binding sites of the homologous agonist structures GR4 (dexamethasone), PR (PDB 1A28
[PDB]
, progesterone), and AR (PDB 1I37
[PDB]
, dihydrotestosterone) (Fig. 5) shows that the A-ring side of the steroid hormone is situated in the most conserved protein side chain environment, with Phe-623, Arg-611, and Gln-570, respectively, at identical positions (taking the error of the models into consideration). All these ligands are also very similar in their A- and B-rings. On the D-ring side, on the other hand, unique features exist between different steroid ligands consistent with larger differences between corresponding cognate receptors in the D-ring-harboring part of the ligand-binding pocket. The largest variation in structure between the three receptor LBDs is seen for Gln-642 (Leu-797 in PR, Gln-783 in AR). In GR, Gln-642 makes a hydrogen bond to the 17-hydroxy group. The position of the side chains of the three other residues in GR-LBD that bind to the C/D-ring of the steroid (Asn-564, 11
-hydroxy; Cys-736, 20-keto; Thr-739, 21-hydroxy) are relatively well conserved between the three structures. Thus, Gln-642 appears to play a unique role in steroid recognition.
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We present three different structures of GR complexed with RU-486. GR1 represents the classic antagonist structure where helix 12 stacks into the N-terminal part of the coactivator pocket (Fig. 6). This agrees with what is seen in other nuclear receptor antagonist complexes (26). In GR2 the C-terminal is truncated by enterokinase (amino acids 743777). GR3 reveals major conformational changes. Helix 12 together with the former end of helix 11 and the inter-helix loop stretch out to bind into the exposed agonist and coactivator surface of a neighboring molecule (Fig. 2a). The first part of the loop between helices 11 and 12 (amino acids 740 746) forms a short helix stretch, helix 11a. Then the last nine C-terminal residues (amino acids 768 776) finally return to the same position as seen in the agonistic structure GR4 as well as in the PR agonist structure (8). Helix 12 is restrained by the C-terminal tail, which anchors to GR and limits the flexibility of helix 12. The GR3 structure is also restrained by the formation of the intermolecular cystine. All these structures together reveal an extremely flexible protein capable of adapting its interactions with other proteins in number of different ways. The structures also suggest that there are several ways to antagonize GR.
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FOOTNOTES |
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* The GR1 and GR2 data were collected at beamline 17-ID in the facilities of the Industrial Macromolecular Crystallography Association Collaborative Access Team at the Advanced Photon Source. These facilities are supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Illinois Institute of Technology (IIT), executed through the IIT Center for Synchrotron Radiation Research and Instrumentation. Use of the Advanced Photon Source was supported by the United States Department of Energy, Basic Energy Sciences, Office of Science under Contract W-31-109-Eng-38. GR3 and GR4 data were collected at beamline ID14-4, European Synchrotron Radiation Facility, Grenoble, France. 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.
To whom correspondence should be addressed. E-mail: bjorn.kauppi{at}karobio.se.
1 The abbreviations and trivial names used are: GR, glucocorticoid receptor; AR, androgen receptor; ER, estrogen receptor; LBD, ligand-binding domain; NR, nuclear receptor; PR, progesterone receptor.
2 A. Pike, York, England, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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