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【关键词】 Structural
Spirolactones are potent antagonists of the mineralocorticoid receptor (MR), a ligand-induced transcription factor belonging to the nuclear receptor superfamily. Spirolactones are synthetic molecules characterized by the presence of a C17 -lactone, which is responsible for their antagonist character. They harbor various substituents at several positions of the steroid skeleton that modulate their potency in ways that remain to be determined. This is particularly obvious for C7 substituents. The instability of antagonist-MR complexes makes them difficult to crystallize. We took advantage of the S810L activating mutation in MR (MRS810L), which increases the stability of ligand-MR complexes to crystallize the ligand-binding domain (LBD) of MRS810L associated with 7-acetylthio-17-hydroxy-3-oxopregn-4-en-21-carboxylic acid -lactone (SC9420), a spirolactone with a C7 thioacetyl group. The crystal structure makes it possible to identify the contacts between SC9420 and MR and to elucidate the role of Met852 in the mode of accommodation of the C7 substituent of SC9420. The transactivation activities of MRS810L/Q776A, MRS810L/R817A, and MRS810L/N770A reveal that the contacts between SC9420 and the Gln776 and Arg817 residues are crucial to maintaining MRS810L in its active state, whereas the contact between SC9420 and the Asn770 residue contributes only to the high affinity of SC9420 for MR. Moreover, docking experiments with other C7-substituted spirolactones revealed that the MRS810L-activating potency of spirolactones is linked to the ability of their C7 substituent to be accommodated in LBD. It is remarkable that the MRS810L-activating and MRWT-inactivating potencies of the C7-substituted spirolactones follow the same order, suggesting that the C7 substituent is accommodated in the same way in MRS810L and MRWT. Thus, the MRS810L structure may provide a powerful tool for designing new, more effective, MR antagonists.
Aldosterone plays a major role in regulating sodium and potassium homeostasis in tight epithelia, including the distal tubule of the kidney, the distal colon, and the salivary and sweat glands (Horisberger and Rossier, 1992; Bonvalet, 1998). It may also exert pathophysiological effects in nonepithelial target tissues, such as the adipose tissue (Penfornis et al., 2000) and the cardiovascular system, where it contributes to controlling blood pressure and is implicated in some disorders, such as hypertension (Rossi et al., 2005).
Aldosterone exerts its effects by binding to the mineralocorticoid receptor (MR), a ligand-activated transcription factor that is a member of the nuclear receptor superfamily (Mangelsdorf et al., 1995; Gronemeyer et al., 2004). Aldosterone-dependent activation of gene transcription is thought to be a multistep process. In its ligand-free state, MR is predominantly located in the cytoplasm, where it is associated with a protein complex, including the 90-kDa heat shock protein. When aldosterone binds to MR this induces a receptor conformation change that in turn leads to the dissociation of the associated proteins, the transfer of the complex into the nucleus, and the subsequent recruitment of transcriptional coactivators (Trapp et al., 1995; Couette et al., 1996; Fejes-Tóth et al., 1998; Hellal-Levy et al., 2000; Hultman et al., 2005).
Spirolactones are MR antagonists that have been widely used for the past 30 years in the treatment of sodium-retaining states and as antihypertensive agents. They improve survival in heart failure, and they have beneficial effects in preventing the development of cardiac fibrosis and renal damage in patients with essential hypertension (Pitt et al., 1999, 2003; Garthwaite and McMahon, 2004). Spirolactones are synthetic molecules with a C17 -lactone that is responsible for their antagonist activity and various other substituents on the steroid skeleton that modulate their potency (Corvol et al., 1977; Nickisch et al., 1985; de Gasparo et al., 1987; Elger et al., 2003; Fagart et al., 2005a); this is particularly evident in the case of the C7 substituents. SC9420, a member of the spirolactone family, characterized by the presence of a C7 thioacetyl group, and RU26752, which has a C7 propyl group, more potently inactivate MRWT than mexrenone, which harbors a C7 carboxymethyl ester group, or canrenone, which has no C7 substituent (Fagart et al., 2005a). In this study, we set out to elucidate how the C7 substituent regulates the antagonist potency of spirolactones.
The crystal structure of the ligand-binding domain (LBD) of MR when it is associated with an agonist is now available (Bledsoe et al., 2005; Fagart et al., 2005b; Li et al., 2005). It is likely that the high stability of the agonist-MR complexes has facilitated their purification and crystallization. In contrast, the low stability of the antagonist-MR complexes (Fagart et al., 1998) has made it impossible to determine the structure of the MR-LBD associated with an antagonist ligand. It is noteworthy that the S810L mutation, which is responsible for a severe familial form of hypertension, increases the stability of ligand-MR complexes, and switches SC9420 from an antagonist to an agonist (Geller et al., 2000; Rafestin-Oblin et al., 2003; Pinon et al., 2004). These properties have made it possible to determine the crystal structure of MRS810L-LBD associated with SC9420 (Bledsoe et al., 2005). Unfortunately, the acetyl moiety of the C7 side chain is missing in this structure, and the report of Bledsoe et al. (2005) makes it clear that the ligand density ends after the sulfur atom. To characterize the anchoring of the C7 substituent of SC9420 and to clarify how the C7 substituent of spirolactones can modulate potency, we solved the crystal structure of MRS810L-LBD associated with SC9420 using the same experimental conditions that we used to determine the structure of MRS810L-LBD associated with progesterone and deoxycorticosterone (Fagart et al., 2005b). We also elucidated the mechanism by which MRS810L is activated by spirolactones and identified the contacts that modulate ligand affinity.
Chemicals. Aldosterone (4-pregnen-11,21-diol-18-al-3,20-dione), progesterone (4-pregnen-3,20-dione), and SC9420 were purchased from Sigma-Aldrich (St Louis, MO). RU26752 was kindly provided by Sanofi-Aventis (Paris, France). Canrenone was provided by Pfizer Inc. (New York, NY). Mexrenone was a gift from G. Auzou (Institut National de la Santé et de la Recherche Médicale U540, Montpellier, France). 18-oxo-18-Vinylprogesterone (18-vinyl-4-pregnen-3,18,20-trione; 18OVP) was a gift from A. Marquet (Paris, France). All other products used in the biochemical studies were purchased from Sigma-Aldrich (St Louis, MO).
Expression Vectors. The expression vector pchMRWT contains the entire coding sequence of wild-type human MR (Fagart et al., 1998). The expression vectors pchMRN770A, pchMRS810L, pchMRS810L/Q776A, and pchMRS810L/R817A contain the coding sequences of mutant MRN770A, MRS810L, MRS810L/Q776A, and MRS810L/R817A, respectively (Fagart et al., 1998, 2005b; Rafestin-Oblin et al., 2003). The expression vector pchMRS810L/N770A was created by cutting out the Bpu1102I-AflII fragment from pchMRS810L and inserting it into pchMRN770A. The plasmid pcgal contains the coding sequence for the -galactosidase (Fagart et al., 2005a). The plasmid pFC31Luc contains the MMTV promoter that drives the luciferase gene (Gouilleux et al., 1991). The expression vector pMRLBDL810 contains the coding sequence for the fusion protein between GST and MR-LBD that harbors the S810L and C910A mutations (Fagart et al., 2005b).
Protein Expression and Purification. Fermentation using the BL21 CodonPlus (DE3) RIL strain from Stratagene (Amsterdam, The Netherlands) transformed with the pMRLBDL810 vector was carried out in the presence of 100 µM SC9420. Expression was induced by incubating with 200 µM isopropyl--D-thiogalactoside for 16 h at 15°C. After centrifuging, the bacteria were disrupted by sonication in TENG buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, and 0.1% n-octyl--glucoside) supplemented with 100 µM SC9420. The lysate was clarified and loaded onto a GSTrap column (GE Healthcare, Les Ulis, France). The fusion protein was eluted with 15 mM reduced glutathione in the TENG buffer. After diluting the eluate to a protein concentration of 1 mg/ml, the fusion protein was cleaved by exposing to thrombin protease (20 units/mg of fusion protein) overnight at 4°C. The protein mixture was diluted a further 5-fold in a HENG buffer (10 mM HEPES, pH 6.8, 10% glycerol, and 0.1% n-octyl--glucoside) supplemented with 100 µM SC9420, loaded onto a sulfoxide column (SP XL from GE Healthcare), and eluted with a gradient of 0 to 500 mM NaCl in the HENG buffer. The fractions containing the LBD were pooled and concentrated to a protein concentration of 7 mg/ml.
Crystallization and Data Collection. Crystals were grown over few days at room temperature in hanging drops containing 1 µlof protein solution and 1 µl of well buffer (100 mM HEPES, pH 6.8, 230 mM NaCl, and 25% PEG 4000). Before data collection, the crystals were flash-frozen in liquid nitrogen without adding any cryoprotecting agent. Diffraction data were collected to a resolution of 2.29 Å, at a temperature of –80.1°C and at a wavelength of 0.979707 Å on the FIP-BM30A beamline at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) using a MarCCD detector. The data set was integrated and scaled using XDS (Kabsch, 1993).
Structure Determination and Refinement. The crystal structure was determined by molecular replacement, using Phaser (Storoni et al., 2004) with the coordinates of the MRS810L-LBD associated with deoxycorticosterone (PDB ID 1Y9R; Fagart et al., 2005b) as the search model. The complex crystallized in the P32 space group. From molecular replacement, two rotation solutions clearly appeared, and three translation solutions were obtained for each of these rotation solutions, indicating the presence of six molecules in the asymmetric unit. Several rounds of manual rebuilding using the SigmaA weighted 2 Fo – Fc electron density maps, followed by simulated annealing and individual isotropic B factor refinements were performed using CNS (Brünger et al., 1998). Solvent molecules were located in a Fo – Fc map contoured at 2. The final R and R-free values were 24 and 26.3%, respectively. The final model was validated with PROCHECK (Laskowski et al., 1993); 99.7% of the residues lie within the allowed regions of the Ramachandran Plot.
Determination of the Cavity Volumes. The volume of the ligand-binding cavity of MRS810L, associated with SC9420, and that of MRS810L, associated with progesterone, were calculated using the probe-occupied algorithm of the VOIDOO package (Kleywegt and Jones, 1994). This algorithm uses a probe-sphere with a radius of 1.4 Å. The contacts between the probe-sphere and the van der Waals protein surface delineate the probe-occupied cavity. The volume of the ligand-binding cavity of MRS810L, associated with SC9420, and that of MRS810L, associated with progesterone, were calculated for each monomer of the asymmetric unit. The values reported under Results are the means of the monomer volumes.
Docking Experiments. RU26752 and mexrenone were constructed and minimized using the Insight II package (Accelrys, Cambridge, UK). Both spirolactones were manually docked within the crystal structure of the MRS810L-LBD associated with SC9420 using the O package (Jones et al., 1991).
Cells Culture and Transfection Procedures. HEK 293T cells were cultured in high-glucose-containing Dulbecco's minimal essential medium (Invitrogen, Cergy Pontoise, France), 25 mM HEPES, 2x nonessential amino acids, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, supplemented with 10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere and with 5% CO2. Four hours before transfection, the cells were cultured in the same medium supplemented with 10% charcoal-treated fetal calf serum. Transfections were carried out using the calcium phosphate precipitation method. Cells were transfected with 2 µg of one of the receptor expression vectors (pchMRWT, pchMRN770A, pchMRS810L, pchMRS810L/N770A, pchMRS810L/Q776A, or pchMRS810L/R817A), 7 µg of pFC31Luc and 1 µg of pcgal in 1x HEPES-buffered saline supplemented with 160 mM CaCl2. Twelve hours after transfection, the cells were rinsed with phosphate-buffered saline, trypsinized, and replated in 12-well plates. The steroids to be tested were added to the cells 24 h after seeding. After incubating for 24 h, cell extracts were assayed for luciferase and -galactosidase activities (Herbomel et al., 1984; de Wet et al., 1987). To standardize the transfection efficiency, the relative light units obtained in the luciferase assay were divided by the optical density obtained in the -galactosidase assay. Each point is the mean ± S.E.M. of three separate experiments.
Effects of the C7 Substituent of Spirolactones on the Agonist Activity of MRS810L. It has been reported that SC9420, a spirolactone with a C7 thioacetyl group, activates the MR carrying the S810L mutation (MRS810L) (Geller et al., 2000). We wondered whether spirolactones lacking C7 substituent (canrenone) or harboring a C7 substituent distinct from that of SC9420 (RU26752 and mexrenone; see formulae in Fig. 1) are also able to activate MRS810L. We investigated the ability of these spirolactones to activate MRS810L transiently expressed in HEK293T cells. Transfection assays performed with pchMRS810L revealed that RU26752, which is characterized by having a C7 propyl group, activates MRS810L as potently as SC9420 and has an ED50 value of 10–10 M, close to that of aldosterone (ED50 5 x 10–11 M) (Fig. 2). Mexrenone, with a C7 carboxymethyl ester group, is less potent than SC9420 (ED50 10–9 M) (Fig. 2). Canrenone, which has no C7 substituent, displays lower agonist activity corresponding to 60% of the maximum aldosterone-induced MRS810L activity (Fig. 2) and has no antagonist activity (data not shown). Thus, all the spirolactones tested activate MRS810L, and their potencies depend on the C7 substituents. It is interesting that the MRS810L-activating and MRWT-inactivating (Fagart et al., 2005a) potencies of the C7-substituted spirolactones follow the same order, suggesting that the C7 substituent is accommodated in the same way in MRS810L and MRWT.
Fig. 1. Structural formulae of spirolactones.
Fig. 2. Effect of spirolactones on the MRS810L transactivation activity. HEK 293T cells transiently expressing MRS810L were incubated for 24 h with increasing concentrations (10–11–10–6 M) of aldosterone (ALDO), SC9420, RU26752, mexrenone (MEX), or canrenone (CAN). The transactivation activities of MRS810L were determined from the luciferase activity normalized in terms of the -galactosidase activity. Results are expressed as a percentage of MRS810L activity in response to 10–9 M aldosterone. Values are mean ± S.E.M. of three separate experiments.
Crystal Structure of the Ligand-Binding Domain of MRS810L Associated with SC9420. To identify the accommodation mode of SC9420 within the ligand-binding cavity of MR, we solved the crystal structure of the LBD of MRS810L associated with SC9420. The MRS810L-LBD was expressed as a fusion protein with glutathione transferase (GST) in the presence of a high concentration of SC9420 and then purified according to the protocol described previously (Fagart et al., 2005b). In brief, the fusion protein was purified by affinity chromatography and cleaved by the action of the thrombin protease. The LBD was separated from GST by cation exchange chromatography and then crystallized by the vapor diffusion method. The structure was solved by molecular replacement, using the crystal structure of MRS810L-LBD associated with deoxycorticosterone as a template (PDB ID 1Y9R; Fagart et al., 2005b) and was then refined to 2.3 Å resolution (see Table 1). The complex crystallized in the P32 space group with six molecules in the asymmetric unit (see Table 1). MRS810L-LBD associated with SC9420 is composed of 11 -helices (H1, H3–H12) and two short -sheets, organized into three layers. The quality of the density map made it possible to pinpoint the position of SC9420 accurately in the ligand-binding cavity (Fig. 3A). This cavity is lined by 22 residues, five of which are polar, and three anchor the ligand (Fig. 3B). The C3 ketone function of SC9420 is hydrogen bound to the Gln776 and Arg817 residues and to a water molecule (Fig. 3B). The ketone function of the C17 -lactone of SC9420 establishes a hydrogen bond with the Asn770 residue (Fig. 3B). Seventeen residues contribute to the hydrophobic nature of the binding cavity and stabilize the position of SC9420 through numerous van der Waals contacts (Fig. 3B). The Leu810 residue forms short hydrophobic contacts with the C19 methyl group of SC9420 and with the Gln776 residue (Fig. 3, A and B). The C7 thioacetyl group of SC9420 is clearly defined in the electron density map (Fig. 3A). It is accommodated within a small hydrophobic groove delimited by Ser811 (H5), Leu814 (H5), Leu827 (-turn), Phe829 (-turn), Met845 (H7), Cys849 (H7), Met852 (H7), and Leu938 (H11) (Fig. 3B), where it establishes numerous van der Waals contacts.
TABLE 1 Data collection and refinement statistics
Fig. 3. Crystal structure of the MRS810L-LBD associated with SC9420. A, stereo view of the 2 Fo – Fc electron density map showing SC9420, and the surrounding residues in the MRS810L-LBD. The map was calculated at 2.29 Å and contoured at 1 . This figure was produced using DINO (http://www.dino3d.org). B, diagram showing the interactions between MRS810L-LBD and SC9420. Hydrogen bonds and van der Waals interactions are depicted as solid red arrows and dashed black lines, respectively. W indicates a water molecule.
We next compared the crystal structure of MRS810L, associated with SC9420, with that of MRS810L, associated with progesterone, a ligand with no C7 substituent (PDB ID 1YA3; Fagart et al., 2005b). The orientations of the Ser811, Met845, and Met852 residue side chains of the ligand-binding cavity are modified (compare Fig. 4A and B). The slight changes in the orientation of the Ser811 and the Met845 residue side chains modify neither the van der Waals volume occupied by these residues nor the volume of the ligand-binding cavity. In contrast, changing the orientation of the Met852 residue has a drastic impact. Its side chain adopts a folded-back conformation in the presence of the C7-substituted SC9420 (Fig. 4A) that is different from the extended conformation observed with progesterone, a steroid with no C7 substituent (Fig. 4B). Accordingly, the volume of the ligand-binding cavity of MRS810L associated with SC9420 is larger than that of MRS810L associated with progesterone (499 Å3 versus 406 Å3) (Fig. 4, A and B). Thus, the presence of the C7 thioacetyl group of SC9420 modifies the conformation of the Met852 side chain, creating a small groove within which the C7 substituent is accommodated and establishes numerous contacts.
Fig. 4. Ligand-binding pockets of MRS810L-LBD associated with SC9420 (A) and progesterone (PDB ID 1YA3) (B). The ligand cavities volumes (gray) were calculated by VOIDOO (Kleywegt and Jones, 1994). This figure was produced using DINO (http://www.dino3d.org).
Finally, we compared the crystal structure of MRS810L, associated with SC9420, and that of MRWT, associated with deoxycorticosterone, a molecule that activates both MRWT and MRS810L (PDB ID 2ABI). Superimposing the two structures reveals that the overall organization of the two structures is very similar, the positioning of all 11 helices being the same (Fig. 5A). Superimposing the ligand-binding cavity of MRS810L, associated with SC9420, over that of MRWT, associated with deoxycorticosterone, shows that the Leu810 residue establishes short hydrophobic contacts with the C19 methyl group of SC9420, and with the Gln776 residue (Fig. 5B). The superimposition of the two structures also reveals that the network of contacts, created by the Leu810 residue, does not exist in the MRWT (Fig. 5B). Thus, the S810L mutation does not modify the overall organization of the receptor but does allow additional contacts to occur that stabilize the receptor in its active state.
Fig. 5. Superimposition of the structure of the LBD of MRS810L associated with SC9420 (blue) over that of MRWT, associated with deoxycorticosterone (gray) (PDB ID 2ABI). A, overall organization of the LBDs. B, focus on the ligand-binding cavities. Hydrogen bonds and van der Waals interactions are depicted as dashed green and black lines, respectively. This figure was produced using DINO (http://www.dino3d.org).
C7-Substituted Spirolactones Docking within the MRS810L-LBD. To find out how the C7 side chain of RU26752 and mexrenone can be accommodated within the ligand-binding cavity, we determined the lowest energy conformation of the C7 substituents of spirolactones by performing / rotation searches and docked these molecules within the structure of MRS810L-LBD associated with SC9420. The orientation of the thioacetyl group of SC9420 determined by the lowest energy conformation search is the same as that observed in the crystal structure, suggesting that no modification of the C7 substituent orientation occurs when SC9420 binds to MRS810L (Fig. 6A). The minimized conformations search reveals that the propyl group of RU26752 is oriented in the same way as the thioacetyl group of SC9420, and docking experiments show that it fits well into the groove created by the folded-back conformation of the Met852 residue, where it establishes numerous contacts (Fig. 6B). In contrast, the minimization of mexrenone shows that the orientation of its C7 side chain is different from that of SC9420. The docking experiment of the minimized mexrenone within the crystal structure of MRL810-LBD associated with SC9420 reveals that its C7 substituent is too close to the Phe829 and the Leu938 residues, leading to unfavorable contacts (Fig. 6C). The rotation of the C7 side chain would be possible, but would necessitate energy-consuming adaptation of the receptor and/or of mexrenone. Thus, the accommodation mode of the C7 substituent of spirolactones is directly linked to their structure.
Fig. 6. Spirolactones within the ligand-binding cavity of MRS810L. A, superimposition of the minimized SC9420 (blue) over the SC9420 in the crystal structure (gray) within the ligand-binding cavity of MRS810L. Docking of the lowest energy conformations of RU26752 (B) and mexrenone (C) within the ligand-binding cavity of MRS810L. Van der Waals volumes of the C7 substituent are depicted in red and those of Phe829 and Leu938 in black. The figure was generated using Open PyMol version 0.93.
Activation Mechanism of the MRS810L by Spirolactones. The structure revealed that SC9420 is anchored by several hydrogen bonds, between the C3 ketone function and the Gln776 and the Arg817 residues and between the C17 -lactone and the Asn770 residue. Which contacts are involved in stabilizing spirolactone-MRS810L in its active state? To identify these contacts, we replaced the polar residues Asn770, Gln776, or Arg817 by an alanine within MRS810L and then tested the ability of spirolactones with various C7 substituent to activate the corresponding double-mutant receptors (MRS810L/N770A, MRS810L/Q776A, and MRS810L/R817A). Transfection assays performed with pchMRS810L/Q776A and pchMRS810L/R817A showed that all the spirolactones tested were unable to activate MRS810L/Q776A and MRS810L/R817A (data not shown), which contrasted with their ability to activate MRS810L. The next question was whether spirolactones act as antagonist ligands when bound to MRS810L/Q776A and MRS810L/R817A. To answer this question, HEK 293T cells transiently expressing MRS810L/Q776A or MRS810L/R817A were incubated with aldosterone, in the presence of rising concentrations of spirolactones. Aldosterone has been reported to activate MRS810L/Q776A and MRS810L/R817A with ED50 values of 10–7 and 10–8 M, respectively (Fagart et al., 2005b). RU26752 and SC9420 inhibit the aldosterone-induced transactivation activities of MRS810L/Q776A and MRS810L/R817A in a dose-dependent manner with IC50 values ranging from 1 to 5 x 10–7 M (Fig. 7, A and B). Mexrenone and canrenone also inhibited the aldosterone-induced transactivation activities of MRS810L/Q776A and MRS810L/R817A but with less potency, their IC50 values ranging from 5 to 10 x 10–6 M (Fig. 7, A and B). Thus, the Q776A and the R817A mutations within MRS810L abolish the agonist character that spirolactones display when bound to the MRS810L. In addition, the potency of spirolactones for inactivating MRS810L/Q776A and MRS810L/R817A follows the same order as that for activating MRS810L (RU26752 > SC9420 > mexrenone > canrenone). Overall, these findings indicate that the S810L mutation stabilizes the spirolactone-receptor complexes in their active state, by reinforcing their contacts with the Gln776 and the Arg817 residues without modifying their potencies.
Fig. 7. Transactivation properties of MRS810L/Q776A and MRS810L/R817A in response to spirolactones. HEK 293T cells transiently expressing MRS810L/Q776A (A) and MRS810L/R817A (B) were incubated for 24 h with 10–7 M aldosterone in the absence (100% agonist activity) or presence of increasing (10–8 Mto10–5 M) concentrations of SC9420, RU26752, mexrenone (MEX), and canrenone (CAN). Transactivation activities of mutant MRs were determined from the luciferase activity normalized in terms of the -galactosidase activity. Values are mean ± S.E.M. of three separate experiments.
We then investigated the role of the Asn770 residue in MRS810L activation by spirolactones. Progesterone activated MRS810L/N770A with an ED50 value of 5 x 10–9 M, but aldosterone did not (data not shown). We therefore used progesterone as an agonist ligand in the transfection assays performed with pchMRS810L/N770A. At a concentration of 10–5 M, RU26752 induced 60% of the maximum progesterone-induced MRS810L/N770A activity (Fig. 8A). At the same concentration, SC9420, mexrenone, and canrenone activate MRS810L/N770A by 13, 10, and 3%, respectively (Fig. 8A). The question then arose as to whether spirolactones would display antagonist properties when bound to MRS810L/N770A. Transfection assays performed with pchMRS810L/N770A revealed that at a concentration of 10–5 M, SC9420 inhibited progesterone-induced MRS810L/N770A activity by 75%, whereas RU26752, mexrenone, and canrenone antagonized the effects of progesterone by only 11, 29, and 27%, respectively (Fig. 8B). Spirolactones modified the transactivation properties of MRS810L/N770A only at a high concentration (10–5 M). As the N770A mutation within MRS810L dramatically reduced the ability of spirolactones both to activate and inactivate the receptor, it seems likely that it must play a key role in the affinity of spirolactones for MRS810L.
Fig. 8. Transactivation properties of MRS810L/N770A in response to spirolactones. A, HEK 293T cells transiently expressing MRS810L/N770A were incubated for 24 h with 10–7 M progesterone (P) or 10–7 to 10–5 M spirolactones to be tested. Results are expressed as the percentage of the MRS810L/N770A activity in response to 10–7 M progesterone. B, HEK 293T cells transiently expressing MRS810L/N770A were incubated for 24 h with 10–7 M progesterone in the absence (100% agonist activity) or presence of increasing (10–7 M–10–5 M) concentrations of the spirolactones tested. Transactivation activities of MRS810L/N770A were determined from the luciferase activity normalized in terms of the -galactosidase activity. Values are mean ± S.E.M. of three separate experiments.
Role of the Asn770 Residue in MRWT. It has been reported that the antagonist character of spirolactones when bound to the MRWT is due to their inability to establish contact with the Asn770 residue (Fagart et al., 1998). We observed here that the contact between spirolactones and the Asn770 residue is important for their high affinity for MRS810L. This led us to wonder whether the Asn770 residue also contributes to the high affinity of spirolactones for MRWT. The Asn770 residue was replaced by an alanine in the context of the wild-type receptor, and the ability of the corresponding mutant MRN770A to be inactivated by spirolactones was tested and compared with that of MRWT. The synthetic compound 18OVP was used in the transfection assays because it is able to activate both MRWT and MRN770A with the same potency (ED50 values of 5 x 10–8 M) (Souque et al., 1995; Fagart et al., 1998). SC9420, RU26752, mexrenone, and canrenone very potently inhibited the MRWT activity induced by 18OVP (Fig. 9A). Complete inhibition was observed for a concentration of spirolactones of 10–6 M (Fig. 9A). After the N770A mutation within MRWT, all the spirolactones retained their antagonist character, but they displayed lower potency (Fig. 9B). At 10–6 M, SC9420, mexrenone, and canrenone inhibited the 18OVP-induced MRN770A activity by 20 to 45%, compared with more than 90% inhibition of the MRWT (Fig. 9B). The antagonist potency of RU26752 was also decreased but to a lesser extent. Indeed, at a concentration of 10–7 M, RU26752 inhibited the 18OVP-induced activity of MRWT and MRN770A by 90 and 40%, respectively (Fig. 9B). Thus, it can be suggested that the Asn770 residue may play a key role in the affinity of spirolactones for MRWT.
Fig. 9. Transactivation properties of MRWT and MRN770A in response to spirolactones. HEK 293T cells transiently expressing MRWT (A) or MRN770A (B) were incubated for 24 h with 10–7 M 18OVP in the absence (100% agonist activity) or presence of increasing (10–7 M–10–5 M) concentrations of the spirolactones tested. Transactivation activities of MRs were determined from the luciferase activity normalized in terms of the -galactosidase activity. Values are mean ± S.E.M. of three separate experiments.
The present study shows that spirolactones activate the MR harboring the S810L mutation, which is responsible for hypertension, whereas they act as antagonists when bound to the wild-type receptor. It also indicates that the potencies of spirolactones in activating MRS810L and inactivating MRWT follow the same order, allowing us to propose that the contacts involved in MRS810L-activation by spirolactones may be different from those that modulate their potency.
The understanding of the activation mechanism of nuclear receptors has been greatly improved by crystallographic studies of LBDs in their inactive and active states. The LBD of nuclear receptors that surrounds the ligand-binding cavity is rather dynamic and exhibits some of the properties of a molten globule in the absence of ligand (Nagy and Schwabe, 2004). Binding a ligand compacts the LBD by establishing many polar and hydrophobic contacts. Some of these are involved in the stability of the ligand-receptor complex, and others are required to stabilize the complex in its active state, facilitating the recruitment of transcriptional coactivators (Nagy and Schwabe, 2004). Several structures of the LBD of MR in its agonist conformation are now available (Bledsoe et al., 2005; Fagart et al., 2005b; Li et al., 2005). In contrast, no structure of MR in its apo and antagonist conformation is yet available, making it impossible to identify the conformational changes that take place in response to agonist or antagonist binding. Nevertheless, a few years ago, it was shown that the contact between the Asn770 residue of MRWT and the C21 hydroxyl function of aldosterone, or the C11 hydroxyl function of 11-hydroxyprogesterone, is responsible for the agonist character of these molecules (Fagart et al., 1998; Rafestin-Oblin et al., 2002). In contrast, the MR antagonists, such as progesterone or spirolactones, are unable to contact the Asn770 residue (Fagart et al., 1998). Thus, the mechanism by which antagonist ligands inactivate the MR is based on the instability of the antagonist-MR complexes, rather than on the ability of the antagonist ligand to stabilize the MR in an inactive state, favoring the recruitment of corepressors (Fagart et al., 1998).
In this study, transactivation experiments revealed that spirolactones activate MR, harboring the S810L mutation (MRS810L). These results raised the question of how the S810L mutation modifies spirolactone-receptor contacts, allowing MRS810L to be maintained in its active state. The crystal structure of the MRS810L-LBD associated with SC9420 reported here reveals that the domain is composed of 11 -helices (H1, H3–H12) and two short -sheets, organized into three layers. The H12 helix is folded back toward the core of the domain, closing the ligand-binding pocket. The C-terminal extension is anchored to the region delineated by H8, H9, and H10 helices by numerous van der Waals contacts and by hydrogen bonds. This overall organization is similar to that of MR in its active state (Bledsoe et al., 2005; Fagart et al., 2005b; Li et al., 2005). Thus, the S810L mutation does not modify the positioning of the helices of the LBD. The H12 helix, which plays a crucial role in the activation process of the steroid receptors, adopts the same position in MRWT and MRS810L. This finding suggests that agonist binding to MRWT and MRS810L leads to the recruitment of transcriptional coactivators that occurs in a similar way.
The crystal structure of MRS810L-LBD associated with SC9420 reveals that the Leu810 residue establishes short hydrophobic contacts with the C19 methyl of SC9420 and the Gln776 residue, which do not exist in MRWT. It also reveals that SC9420 is anchored by the Gln776 and the Arg817 residues, and also by the Asn770 residue. This last contact was remarkable, because MRWT-inactivation by spirolactones is based on the absence of contact between the Asn770 residue and spirolactones (Fagart et al., 1998). This led us to wonder about the contribution made by each of the contacts between spirolactones and the polar residues Asn770, Gln776, and Arg817 to the process of MRS810L activation. Mutagenesis analysis revealed that the ability of spirolactones to modulate the MRS810L-activity is dramatically reduced by the N770A mutation. Thus, within MRS810L, the contact between the Asn770 residue and spirolactones is not crucial for stabilizing MRS810L in its active state, but it does play a key role in the affinity of spirolactones. It is noteworthy that steroids harboring a C11 hydroxyl group, such as 11-hydroxyprogesterone and cortisol, or a C11-C18 hemiketal group, as aldosterone, are not able to activate MRS810L/N770A to a significant degree (Bledsoe et al., 2005). Only progesterone and deoxycorticosterone, which have no C11 substitution, are able to activate MRS810L/N770A (Bledsoe et al., 2005). Overall, these results show clearly that, in the context of MRS810L, the anchoring of steroids having a C11 hydroxyl function or a C17 -lactone involves the Asn770 residue.
We wondered whether the two other polar residues Gln776 and Arg817, which anchor the C3 ketone function of spirolactones, play any role in stabilizing spirolactone-MRS810L complexes in their active state. The replacement of the Gln776 or Arg817 residue by an alanine within MRS810L abolishes their agonist character and restores the antagonist character that spirolactones display when bound to MRWT. Furthermore, the potencies of spirolactones in inactivating MRWT, MRS810L/Q776A, and MRS810L/R817A and activating MRS810L follow the same order. This suggests that the contacts involving the Gln776 and Arg817 residues play a minor role in modulating the affinity of spirolactones for MRS810L but are crucial for the activation of MRS810L by spirolactones. Thus, the S810L mutation within MR reinforces the contacts between the C3 ketone function of spirolactones and the Gln776 and the Arg817 residues; each of these contacts becomes crucial for stabilizing the spirolactone-MRS810L complexes in their active state. The activation of MRS810L by progesterone also requires strong stabilizing contacts implicating the Gln776 and the Arg817 residues, whereas these contacts are dispensable for the activation of MRS810L by C21-hydroxylated compounds, such as aldosterone and deoxycorticosterone (Fagart et al., 2005b). Thus, the Gln776 and the Arg817 residues are implicated in the mechanism of MRS810L activation by steroids harboring a ketone function at the C3 position, but with no C21 hydroxyl function. These findings are consistent with the antagonist property of the synthetic ligand 5-pregnane-20-one that is unable to contact the Gln776 and the Arg817 residues because of the absence of a C3 ketone function (Pinon et al., 2004).
We observed that the MRS810L-activating potency of spirolactones depends on their C7 substituents. The structure reported here reveals that the side chain of the Met852 residue, a residue facing the C7 substituent of SC9420, adopts a folded-back conformation. In all the currently reported structures of MR-LBD associated with a ligand without the C7 substituent, the side chain of the Met852 residue adopts an extended conformation (Bledsoe et al., 2005; Fagart et al., 2005b; Li et al., 2005). This makes it likely that molecular adaptation of the side chain of the Met852 residue may be required to accommodate ligands with a C7 substituent. The folded-back conformation of the Met852 residue side chain creates a small groove surrounded by several hydrophobic residues. The C7 thioacetyl group of SC9420 fits well into this groove, where it makes numerous stabilizing van der Waals contacts. These contacts are responsible for the high MRS810L-activating potency of SC9420. Docking experiments revealed that the C7 propyl group of RU26752 is accommodated in a similar way to the C7 thioacetyl group of SC9420, whereas the C7 carboxymethyl ester group of mexrenone induces steric hindrance with the Phe829 residue. Thus, the accommodation of the C7 substituent within the ligand-binding cavity correlates well with the MRS810L-activating potency of C7-substituted spirolactones.
In conclusion, mutagenesis analyses based on the crystal structure of MRS810L-LBD associated with SC9420, combined with docking experiments, make it possible to distinguish the residues responsible for the MRS810L activation from those that modulate the ligands affinity. The contacts involving the Gln776 and the Arg817 residues are crucial for the activation of MRS810L by spirolactones, whereas the Asn770 and the Met852 residues are key modulators of the affinity of spirolactones for MRS810L. Another important conclusion of the study is that spirolactones also contact the Asn770 residue in MRWT. This contact is not strong enough to stabilize the complex in its active state but is involved in the affinity of spirolactones for MRWT. Because the MRS810L-activating potencies of spirolactones correlate with their potencies in inactivating MRWT, it can be surmised that the C7 substituents are accommodated in same way in MRWT as in MRS810L. Thus, the crystal structure of MRS810L may provide a powerful tool for designing new, more effective, C7-substituted MR antagonists.
Acknowledgements
We thank M. Pirocchi and J.-L. Ferrer from the FIP-BM30A beamline at the European Synchrotron Radiation Facilities for assistance with data collection. We are also grateful to H. Richard-Foy and F. Gouilleux for providing plasmid pFC31Luc. We also thank colleagues for their critical reading of the manuscript.
ABBREVIATIONS: SC9420, 7-acetylthio-17-hydroxy-3-oxopregn-4-en-21-carboxylic acid -lactone (spironolactone); RU26752, 7-propyl-17-hydroxy-3-oxopregn-4-en-21-carboxylic acid -lactone; LBD, ligand-binding domain; 18OVP, 18-oxo-18-vinylprogesterone; GST, glutathione transferase; PDB, Protein Data Bank; HEK, human embryonic kidney; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor.
【参考文献】
Bledsoe RK, Madauss KP, Holt JA, Apolito CJ, Lambert MH, Pearce KH, Stanley TB, Stewart EL, Trump RP, Willson TM, and Williams SP (2005) A ligand-mediated hydrogen bond network required for the activation of the mineralocorticoid receptor. J Biol Chem 280: 31283–31293.[Abstract/Free Full Text]
Bonvalet JP (1998) Regulation of sodium transport by steroid hormones. Kidney Int Suppl 65: S49–S56.
Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, et al. (1998) Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: 905–921.
Corvol P, Claire M, Rafestin-Oblin ME, Michaud A, Roth-Meyer C, and Menard J (1977) Spirolactones: clinical and pharmacologic studies. Adv Nephrol Necker Hosp 7: 199–215.
Couette B, Fagart J, Jalaguier S, Lombes M, Souque A, and Rafestin-Oblin ME (1996) Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure. Biochem J 315: 421–427.
de Gasparo M, Joss U, Ramjoue HP, Whitebread SE, Haenni H, Schenkel L, Kraehenbuehl C, Biollaz M, Grob J, and Schmidlin J (1987) Three new epoxyspirolactone derivatives: characterization in vivo and in vitro. J Pharmacol Exp Ther 240: 650–656.[Abstract/Free Full Text]
de Wet JR, Wood KV, Deluca M, Helinski DR, and Subramani S (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 725–737.[Abstract/Free Full Text]
Elger W, Beier S, Pollow K, Garfield R, Shi SQ, and Hillisch A (2003) Conception and pharmacodynamic profile of drospirenone. Steroids 68: 891–905.
Fagart J, Wurtz JM, Souque A, Hellal-Levy C, Moras D, and Rafestin-Oblin ME (1998) Antagonism in the human mineralocorticoid receptor. EMBO J 17: 3317–3325.
Fagart J, Seguin C, Pinon GM, and Rafestin-Oblin ME (2005a) The Met852 residue is a key organizer of the ligand-binding cavity of the human mineralocorticoid receptor. Mol Pharmacol 67: 1714–1722.[Abstract/Free Full Text]
Fagart J, Huyet J, Pinon GM, Rochel M, Mayer C, and Rafestin-Oblin ME (2005b) Crystal structure of a mutant mineralocorticoid receptor responsible for hypertension. Nat Struct Mol Biol 12: 554–555.
Fejes-Tóth G, Pearce D, and Naray-Fejes-Toth A (1998) Subcellular localization of mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists. Proc Natl Acad Sci U S A 95: 2973–2978.[Abstract/Free Full Text]
Garthwaite SM and McMahon EG (2004) The evolution of aldosterone antagonists. Mol Cell Endocrinol 217: 27–31.
Geller DS, Farhi A, Pinkerton N, Fradley M, Moritz M, Spitzer A, Meinke G, Tsai FT, Sigler PB, and Lifton RP (2000) Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289: 119–123.[Abstract/Free Full Text]
Gouilleux F, Sola B, Couette B, and Richard-Foy H (1991) Cooperation between structural elements in hormono-regulated transcription from the mouse mammary tumor virus promoter. Nucleic Acids Res 19: 1563–1569.[Abstract/Free Full Text]
Gronemeyer H, Gustafsson JA, and Laudet V (2004) Principles for modulation of the nuclear receptor superfamily. Nat Rev Drug Discov 3: 950–964.
Hellal-Levy C, Fagart J, Souque A, and Rafestin-Oblin ME (2000) Mechanistic aspects of mineralocorticoid receptor activation. Kidney Int 57: 1250–1255.
Herbomel P, Bourachot B, and Yaniv M (1984) Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39: 653–662.
Horisberger JD and Rossier BC (1992) Aldosterone regulation of gene transcription leading to control of ion transport. Hypertension 19: 221–227.[Abstract/Free Full Text]
Hultman ML, Krasnoperova NV, Li S, Du S, Xia C, Dietz JD, Lala DS, Welsch DJ, and Hu X (2005) The ligand-dependent interaction of mineralocorticoid receptor with coactivator and corepressor peptides suggests multiple activation mechanisms. Mol Endocrinol 19: 1460–1473.[Abstract/Free Full Text]
Jones TA, Zou JY, Cowan SW, and Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47: 110–119.
Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Crystallogr 26: 795–800.
Kleywegt GJ and Jones TA (1994) Detection, delineation, measurement and display of cavities in macromolecular structures. Acta Crystallogr D Biol Crystallogr 50: 178–185.
Laskowski RA, MacArthur MW, Moss DS, and Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26: 283–291.
Li Y, Suino K, Daugherty J, and Xu HE (2005) Structural and biochemical mechanisms for the specificity of hormone binding and coactivator assembly by mineralocorticoid receptor. Mol Cell 19: 367–380.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, et al. (1995) The nuclear receptor superfamily: the second decade. Cell 83: 835–839.
Nagy L and Schwabe JW (2004) Mechanism of the nuclear receptor molecular switch. Trends Biochem Sci 29: 317–324.
Nickisch K, Bittler D, Casals-Stenzel J, Laurent H, Nickolson R, Nishino Y, Petzoldt K, and Wiechert R (1985) Aldosterone antagonists. 1. Synthesis and activities of 6 beta,7 beta:15 beta,16 betadimethylene steroidal spirolactones. J Med Chem 28: 546–550.
Penfornis P, Viengchareun S, Le Menuet D, Cluzeaud F, Zennaro MC, and Lombes M (2000) The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes. Am J Physiol Endocrinol Metab 279: E386–E394.[Abstract/Free Full Text]
Pinon GM, Fagart J, Souque A, Auzou G, Vandewalle A, and Rafestin-Oblin ME (2004) Identification of steroid ligands able to inactivate the mineralocorticoid receptor harboring the S810L mutation responsible for a severe form of hypertension. Mol Cell Endocrinol 217: 181–188.
Pitt B, Zannad F, Remme WJ, Cody R, Castaigne A, Perez A, Palensky J, and Wittes J (1999) The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators. N Engl J Med 341: 709–717.[Abstract/Free Full Text]
Pitt B, Remme W, Zannad F, Neaton J, Martinez F, Roniker B, Bittman R, Hurley S, Kleiman J, and Gatlin M; Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study Investigators (2003) Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. N Engl J Med 348: 1309–1321.[Abstract/Free Full Text]
Rafestin-Oblin ME, Fagart J, Souque A, Seguin C, Bens M, and Vandewalle A (2002) 11beta-hydroxyprogesterone acts as a mineralocorticoid agonist in stimulating Na+ absorption in mammalian principal cortical collecting duct cells. Mol Pharmacol 62: 1306–1313.[Abstract/Free Full Text]
Rafestin-Oblin ME, Souque A, Bocchi B, Pinon G, Fagart J, and Vandewalle A (2003) The severe form of hypertension caused by the activating S810L mutation in the mineralocorticoid receptor is cortisone related. Endocrinology 144: 528–533.[Abstract/Free Full Text]
Rossi G, Boscaro M, Ronconi V, and Funder JW (2005) Aldosterone as a cardiovascular risk factor. Trends Endocrinol Metab 16: 104–107.
Souque A, Fagart J, Couette B, Davioud E, Sobrio F, Marquet A, and Rafestin-Oblin ME (1995) The mineralocorticoid activity of progesterone derivatives depends on the nature of the C18 substituent. Endocrinology 136: 5651–5658.
Storoni LC, McCoy AJ, and Read RJ (2004) Likelihood-enhanced fast rotation functions. Acta Crystallogr D Biol Crystallogr 60: 432–438.
Trapp T and Holsboer F (1995) Ligand-induced conformational changes in the mineralocorticoid receptor analyzed by protease mapping. Biochem Biophys Res Commun 215: 286–291.
作者单位：Institut National de la Santé et de la Recherche Médicale, U773, Centre de Recherche Biomédicale Bichat Beaujon CRB3, BP 416, Paris, France; and Université Paris 7 Denis Diderot, site Bichat, BP 416, Paris, France