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Insights into the Mechanism of Partial Agonism

CRYSTAL STRUCTURES OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ LIGAND-BINDING DOMAIN IN THE COMPLEX WITH TWO ENANTIOMERIC LIGANDS*
  • Giorgio Pochetti
    Correspondence
    To whom correspondence may be addressed: Istituto di Cristallografia, Sede di Monterotondo, Area della Ricerca ROMA 1, CNR, Via Salaria Km 29,300, 00016 Monterotondo Stazione, Roma, Italia. Tel.: 39-06-90672633; Fax: 39-06-90672630
    Footnotes
    Affiliations
    Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016 Monterotondo Stazione, Roma, Italia
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  • Cristina Godio
    Footnotes
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Nico Mitro
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Donatella Caruso
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Andrea Galmozzi
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Samuele Scurati
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Fulvio Loiodice
    Affiliations
    Dipartimento Farmaco-Chimico, Università degli Studi di Bari, 70125 Bari, Italia
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  • Giuseppe Fracchiolla
    Affiliations
    Dipartimento Farmaco-Chimico, Università degli Studi di Bari, 70125 Bari, Italia
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  • Paolo Tortorella
    Affiliations
    Dipartimento Farmaco-Chimico, Università degli Studi di Bari, 70125 Bari, Italia
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  • Antonio Laghezza
    Affiliations
    Dipartimento Farmaco-Chimico, Università degli Studi di Bari, 70125 Bari, Italia
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  • Antonio Lavecchia
    Affiliations
    Dipartimento di Chimica Farmaceutica, Università degli Studi di Napoli, 80131 Napoli, Italia
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  • Ettore Novellino
    Affiliations
    Dipartimento di Chimica Farmaceutica, Università degli Studi di Napoli, 80131 Napoli, Italia
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  • Fernando Mazza
    Affiliations
    Istituto di Cristallografia, Consiglio Nazionale delle Ricerche, Montelibretti, 00016 Monterotondo Stazione, Roma, Italia

    Dipartimento di Chimica, Ingegneria Chimica e Materiali, Università di L'Aquila, 67010 L'Aquila, Italia
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  • Maurizio Crestani
    Correspondence
    To whom correspondence may be addressed: Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Balzaretti 9, 20133 Milano, Italia. Tel.: 39-02-50318393/1; Fax: 39-02-50318391
    Affiliations
    Laboratorio “Giovanni Galli” di Biochimica e Biologia Molecolare dei Lipidi e di Spettrometria di Massa, Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, 20133 Milano, Italia
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  • Author Footnotes
    * This work was supported by Italian Ministry of University and Research Grant Progetti di Ricerca di Interesse Nazionale 2005033023. 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-5.
    1 These two authors contributed equally to this work.
Open AccessPublished:April 02, 2007DOI:https://doi.org/10.1074/jbc.M702316200
      The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose and lipid metabolism. They are activated by natural ligands, such as fatty acids, and are also targets of synthetic antidiabetic and hypolipidemic drugs. By using cell-based reporter assays, we studied the transactivation activity of two enantiomeric ureidofibrate-like derivatives. In particular, we show that the R-enantiomer, (R)-1, is a full agonist of PPARγ, whereas the S-enantiomer, (S)-1, is a less potent partial agonist. Most importantly, we report the x-ray crystal structures of the PPARγ ligand binding domain complexed with the R- and the S-enantiomer, respectively. The analysis of the two crystal structures shows that the different degree of stabilization of the helix 12 induced by the ligand determines its behavior as full or partial agonist. Another crystal structure of the PPARγ·(S)-1 complex, only differing in the soaking time of the ligand, is also presented. The comparison of the two structures of the complexes with the partial agonist reveals significant differences and is suggestive of the possible coexistence in solution of transcriptionally active and inactive forms of helix 12 in the presence of a partial agonist. Mutation analysis confirms the importance of Leu465, Leu469, and Ile472 in the activation by (R)-1 and underscores the key role of Gln286 in the PPARγ activity.
      The peroxisome proliferator-activated receptors (PPARs),
      The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; LBD, ligand-binding domain; RAR, all-trans-retinoic acid receptor; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; r.m.s., root mean square.
      4The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; LBD, ligand-binding domain; RAR, all-trans-retinoic acid receptor; HPLC, high pressure liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; r.m.s., root mean square.
      members of the superfamily of nuclear receptors, are eukaryotic transcription factors that control the expression of genes involved in fatty acid metabolism (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ,
      • Berger J.
      • Moller D.E.
      ). They function as cellular lipid sensors that activate transcription in response to the binding of a cognate ligand, generally fatty acids and their eicosanoids metabolites (
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ). Ligand binding, by promoting the stabilization of the active conformation of the C-terminal activation function-2 helix (H12), triggers the recruitment of co-activator proteins (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ,
      • Nagy L.
      • Schwabe J.W.
      ,
      • Nettles K.W.
      • Greene G.L.
      ) that locally remodel chromatin and activate the cellular transcriptional machinery (
      • Renaud J.P.
      • Moras D.
      ,
      • Desvergne B.
      • Wahli W.
      ).
      The subtype PPARα promotes fatty acid catabolism in the liver and skeletal muscle, whereas PPARγ regulates fatty acid storage in adipose tissues (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ). PPARs are also targets of synthetic hypolipidemic and antidiabetic drugs. In particular, the fibrate class of lipid-lowering drugs (e.g. fenofibrate and gemfibrozil) are PPARα ligands (
      • Issemann I.
      • Prince R.A.
      • Tugwood J.D.
      • Green S.
      ,
      • Rubins H.B.
      • Robins S.J.
      • Collins D.
      • Fye C.L.
      • Anderson J.W.
      • Elam M.B.
      • Faas F.H.
      • Linares E.
      • Schaefer E.J.
      • Schectman G.
      • Wilt T.J.
      • Wittes J.
      ,
      • Gangloff M.
      • Ruff M.
      • Eiler S.
      • Duclaud S.
      • Wurtz J.M.
      • Moras D.
      ). The thiazolidinedione (
      • Lehmann J.M.
      • Moore L.B.
      • Smith-Oliver T.A.
      • Wilkison W.O.
      • Willson T.M.
      • Kliewer S.A.
      ,
      • Hulin B.
      • McCarthy P.A.
      • Gibbs E.M.
      ) antidiabetic agents (rosiglitazone and pioglitazone) are PPARγ agonists whose insulin-sensitizing action is well established (
      • Schoonjans K.
      • Auwerx J.
      ). Selective PPARγ modulators (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ,
      • Kallenberger B.C.
      • Love J.D.
      • Chatterjee V.K.
      • Schwabe J.W.
      ), acting in a cell type-selective manner and having lower adverse effects, such as edema, cardiomegaly, increased adiposity, and weight gain (
      • Kallenberger B.C.
      • Love J.D.
      • Chatterjee V.K.
      • Schwabe J.W.
      ,
      • Yki-Jarvinen H.
      ,
      • Arakawa K.
      • Ishihara T.
      • Aoto M.
      • Inamasu M.
      • Kitamura K.
      • Saito A.
      ), have been identified. These ligands, which may function as partial agonists, provide diminished conformational stability of receptor as opposed to full agonists (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ,
      • Nettles K.W.
      • Greene G.L.
      ,
      • Steinmetz A.C.
      • Renaud J.P.
      • Moras D.
      ). Furthermore, PPAR ligands with a dual activity on both PPARα and PPARγ receptors improve hyperglycemia and dyslipidemic disorders in a coordinate manner (
      • Berger J.P.
      • Akiyama T.E.
      • Meinke P.T.
      ). Such compounds may lead to preferred therapies for diabetes, obesity, or metabolic syndrome. For this reason, the search for dual agonists with lower potency on PPARγ as compared with full agonists, but also active on PPARα, would be desirable.
      We synthesized the two enantiomers of the novel compound, 2-(4-{2-[1,3-benzoxazol-2-yl(heptyl)amino]ethyl}phenoxy)-2-methyl-butanoic acid, a conformationally constrained analogue of the well known PPARα/γ agonist GW2331 (
      • Kliewer S.A.
      • Sundseth S.S.
      • Jones S.A.
      • Brown P.J.
      • Wisely G.B.
      • Koble C.S.
      • Devchand P.
      • Wahli W.
      • Willson T.M.
      • Lenhard J.M.
      • Lehmann J.M.
      ,
      • Takada I.
      • Yu R.T.
      • Xu H.E.
      • Lambert M.H.
      • Montana V.G.
      • Kliewer S.A.
      • Evans R.M.
      • Umesono K.
      ) (Fig. 1). The R-enantiomer, (R)-1, is able to activate both PPARα and PPARγ, showing higher potency on PPARα. The S-enantiomer, (S)-1, displays a lower efficacy toward PPARγ and behaves as a partial agonist of this receptor subtype.
      Figure thumbnail gr1
      FIGURE 1Schematic diagram of GW2331 (racemic mixture), of (R)-1 and (S)-1 enantiomers and of achiral compound 2.
      We also provide a molecular explanation for their different behavior as full and partial agonists of PPARγ by showing the crystal structures of the complexes of these new ligands with PPARγ ligand binding domain (LBD).
      Among the known crystal structures of PPARγ complexed with partial agonists (
      • Ostberg T.
      • Svensson S.
      • Selen G.
      • Uppenberg J.
      • Thor M.
      • Sundbom M.
      • Sydow-Backman M.
      • Gustavsson A.L.
      • Jendeberg L.
      ,
      • Burgermeister E.
      • Schnoebelen A.
      • Flament A.
      • Benz J.
      • Stihle M.
      • Gsell B.
      • Rufer A.
      • Ruf A.
      • Kuhn B.
      • Marki H.P.
      • Mizrahi J.
      • Sebokova E.
      • Niesor E.
      • Meyer M.
      ,
      • Lu I.L.
      • Huang C.F.
      • Peng Y.H.
      • Lin Y.T.
      • Hsieh H.P.
      • Chen C.T.
      • Lien T.W.
      • Lee H.J.
      • Mahindroo N.
      • Prakash E.
      • Yueh A.
      • Chen H.Y.
      • Goparaju C.M.
      • Chen X.
      • Liao C.C.
      • Chao Y.S.
      • Hsu J.T.
      • Wu S.Y.
      ,
      • Oberfield J.L.
      • Collins J.L.
      • Holmes C.P.
      • Goreham D.M.
      • Cooper J.P.
      • Cobb J.E.
      • Lenhard J.M.
      • Hull-Ryde E.A.
      • Mohr C.P.
      • Blanchard S.G.
      • Parks D.J.
      • Moore L.B.
      • Lehmann J.M.
      • Plunket K.
      • Miller A.B.
      • Milburn M.V.
      • Kliewer S.A.
      • Willson T.M.
      ), PPARγ·(S)-1 is the only one in which the ligand interacts directly with the helix 12; the comparison of this structure with that of the complex with the R-enantiomer permits further insights into the molecular basis of partial agonism in PPARs.
      Although the (R)-1 ligand induces the canonical transcriptionally active conformation of helix 12, the PPARγ·(S)-1 complex shows a suboptimal conformation of this helix, which could be responsible for the partial agonist behavior of the S-enantiomer.

      EXPERIMENTAL PROCEDURES

      Materials—Wy-14,643, dexamethasone, and isopropyl-β-d-thiogalactopyranoside were purchased from Sigma. Dulbecco's modified Eagle's medium/F-12 nutrient (1:1) mixture, fetal calf serum, penicillin, and streptomycin sulfate were purchased from Invitrogen. Invitrogen 49653 (rosiglitazone) was obtained by Hefei Scenery Chemical Co. (Hefei, Anhui, China). GW501516 was purchased from Alexis Corp. (Lausen, Switzerland). Synthesis of compounds (R)-1 and (S)-1 started from the optically active 2-(4-bromophenoxy)-2-methyl-butanoic acids (obtained by resolution of the racemic mixture with (+)- or (-)-1-phenylethylamine) and occurred without affecting the chiral center. The enantiomeric excess was determined by HPLC on Chiralcel OD and estimated as 96% for (R)-1 and 97% for (S)-1. The absolute configuration of (R)-1 and (S)-1 was assigned by chemical correlation with the known (R)- and (S)-2-hydroxy-2-methyl-butanoic acids, from which it was possible to prepare (R)- and (S)-2-(4-bromophenoxy)-2-methyl-butanoic acids, respectively. Achiral compound 2 was obtained following the same procedure starting from 2-(4-bromophenoxy)-2-methyl-propanoic acid. All synthetic details will be reported elsewhere.
      F. Loiodice, P. Tortorella, A. Laghezza, G. Fracchiolla, A. Lavecchia, E. Novellino, F. Mazza, G. Pochetti, C. Godio, N. Mitro, D. Caruso, A. Galmozzi, S. Scurati, and M. Crestani, manuscript in preparation.
      The sequence of biotinylated SRC1 (steroid receptor coactivator 1) peptide spanning amino acids 676-700 was Biotin-CPSSHSSLTERHKILHRLLQEGSPSC-COOH (Biopeptide Co., San Diego, CA); europium-labeled anti-histidine antibody was purchased from PerkinElmer Life Sciences; allophycocyanin-labeled streptavidin was from Prozyme (San Leandro, CA).
      Plasmids—The expression plasmids used for transactivation assays contain the yeast Gal4-DNA-binding domain fused to the human PPARα, PPARγ, or PPARδ LBD in the pSG5 vector, and the reporter plasmid (p5xGal4UAS) has five Gal4 response elements upstream to the luciferase gene. The plasmid used for the expression and purification of the ligand binding domain (residues 193-475) of the human PPARγ contains a tag of six histidines and the cleavage site for thrombin protease upstream of the LBD of PPARγ in the pET28 bacterial expression vector. These plasmids were kindly donated by Dr. Krister Bamberg (AstraZeneca, Mölndal, Sweden). The vectors expressing the Gal4 fusion proteins with the ligand binding domains of RARα, 9-cis-retinoic acid receptor α, RAR-related orphan receptor, thyroid hormone receptor α, glucocorticoid receptor, Nurr1, and farnesoid X receptor were generated by fusing the LBDs to the DNA-binding domain of Gal4 and inserting them into pCDNA3 (Invitrogen) by standard cloning techniques. Assays were validated using known ligands. The expression vector for Gal4-hepatocyte nuclear factor 4 LBD was provided by Dr. Iannis Talianidis (Institute of Molecular Biology and Biotechnology Foundation for Research and Technology Hellas, Herakleion, Crete, Greece). Q286G, I472A, L465A, and L469A mutants were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by automated sequencing.
      Cell Culture and Transfections—Human hepatoblastoma cell line HepG2/C3A (American Type Culture Collection, Manassas, VA) was cultured in Dulbecco's modified Eagle's medium/F-12 nutrient (1:1) mixture containing 10% heat-inactivated fetal calf serum, 100 units of penicillin G/ml, and 100 μg of streptomycin sulfate/ml at 37 °C in a humidified atmosphere of 10% CO2. For transactivation assays, 105 cells/well were seeded in 24-well plates in triplicate, and transfections were performed with a modification of the calcium-phosphate method (
      • De Fabiani E.
      • Mitro N.
      • Gilardi F.
      • Caruso D.
      • Galli G.
      • Crestani M.
      ). After transfection, cells were treated with the indicated ligands for 20 h. Luciferase activity in cell extracts was determined with a luciferase detection kit (Promega, Milan, Italy) using a luminometer (Sirius, Berthold Detection Systems, Pforzheim, Germany). β-Galactosidase activity was determined as previously described (
      • De Fabiani E.
      • Mitro N.
      • Gilardi F.
      • Caruso D.
      • Galli G.
      • Crestani M.
      ). Each experiment was repeated three times.
      Protein Expression and Purification—The LBD of human PPARγ was expressed as N-terminal His-tagged protein using a pET28 vector and purified as previously described (
      • Cronet P.
      • Petersen J.F.
      • Folmer R.
      • Blomberg N.
      • Sjoblom K.
      • Karlsson U.
      • Lindstedt E.L.
      • Bamberg K.
      ). In brief, freshly transformed E. coli BL21 DE3 were grown in LB medium with 30 μg of kanamycin/ml at 37 °C to an OD of 0.6. The culture was then induced with 0.1 mm isopropyl-β-d-thiogalactopyranoside and further incubated at 18 °C for 20 h. Cells were harvested and resuspended in a 20 ml/liter culture of Buffer A (20 mm Tris, 150 mm NaCl, 10% glycerol, 1 mm Tris 2-carboxyethylphosphine HCl, pH 8) in the presence of protease inhibitors (Complete Mini EDTA-free; Roche Applied Science). Cells were sonicated, and the soluble fraction was isolated by centrifugation (35,000 × g for 45 min). The supernatant was loaded onto an Ni2+-nitrilotriacetic acid column (GE Healthcare) and eluted with a gradient of imidazole 0-300 mm in Buffer A with a PU980 HPLC system (Jasco, Lecco, Italy). The fractions containing the protein were collected, quantitated with a Bradford assay, and analyzed on 12% SDS-PAGE (supplemental Fig. 1A). The protein was then dialyzed over Buffer C (20 mm Tris, 20 mm NaCl, 10% glycerol, 1 mm Tris 2-carboxyethylphosphine HCl, pH 8) to remove imidazole, and it was cleaved with thrombin protease (GE Healthcare) (10 units/mg) at room temperature for 2 h. The cleavage was monitored by SDS-PAGE (supplemental Fig. 1A) and by mass spectrometry, and we observed a complete digestion, as assessed by the reduction of 1751 Da of the molecular mass of the recombinant protein, corresponding to the loss of 17 amino acids (supplemental Fig. 1B). The identity of the native and digested protein was determined on the basis of the molecular weight (supplemental Fig. 1B) and of the sequencing of the tryptic peptides obtained by liquid chromatography-electrospray ionization mass spectrometry and tandem mass spectrometry (data not shown) (LTQ; ThermoElectron Co., San Jose, CA), respectively. The digested mixture was reloaded onto an Ni2+-nitrilotriacetic acid column to remove the His tag and the undigested protein. The flow-through was loaded onto a Q-Sepharose HP column (GE Healthcare) and eluted with a gradient of NaCl 0-500 mm in Buffer B (20 mm Tris, 10% glycerol, 1 mm Tris 2-carboxyethylphosphine HCl, pH 8). The protein was then dialyzed over Buffer B and kept frozen in aliquots at a concentration of 1 mg/ml.
      Co-activator Recruitment Assay—His-PPARγ-LBD (20-50 nm) was incubated in black flat-bottom 384-well microplates for 1 h at 37 °C with biotinylated peptide (500 nm), europium-labeled anti-histidine antibody (1 nm), and allophycocyanin-labeled streptavidin (100 nm) in assay buffer (50 mm KCl, 50 mm Tris pH 7.5, 0.1 mg/ml fatty acid-free bovine serum albumin, 1 mm dithiothreitol). Ligands were diluted in Me2SO, starting at the indicated concentration for each ligand. Time-resolved fluorescence was measured on an Analyst GT multimode reader (Molecular Devices, Sunnyvale, CA). Excitation was at 340 nm, and the results are expressed as the ratio of allophycocyanin fluorescence (λ = 665 nm) to europium fluorescence (λ = 620 nm) multiplied by 10,000. The final results represent this ratio minus the background (Me2SO ratio) of six independent experiments. EC50 and Kd values of the ligands were calculated using GraphPad Prism version 4.0c for Macintosh (GraphPad Software, San Diego, CA).
      Competition Binding—The scintillation proximity assay was performed as previously described (
      • Cronet P.
      • Petersen J.F.
      • Folmer R.
      • Blomberg N.
      • Sjoblom K.
      • Karlsson U.
      • Lindstedt E.L.
      • Bamberg K.
      ). Briefly, the assay was performed in 96-well plates (PerkinElmer Life Sciences) using 100 μl of buffer containing 20 mm Tris (pH 7.5), 80 mm NaCl, 2 mm Tris(2-carboxyethyl)phosphine, 0.125% CHAPS, and 10% glycerol. Each well contained 0.1 mg of polylysine-coated yttrium silicate beads (GE Healthcare), 1 μg of His-PPARγ-LBD, and 40 nm [3H]rosiglitazone (American Radiolabeled Chemicals) at 50 Ci/mmol. The amount of protein did not deplete ligand concentration. Cold ligands were tested in 10-point concentration-response curves starting at the indicated concentration. All components were added simultaneously and incubated with gentle shaking for 1 h at room temperature. Scintillation counts were determined in a Microbeta 1450 Wallac Trilux counter (PerkinElmer Life Sciences), reading each well for 1 min. Wells devoid of competitor represented 100% binding. Nonspecific binding was measured by leaving PPARγ protein out of the scintillation proximity assay reaction. Experiments were repeated three times. Ki values of the ligands were calculated using GraphPad Prism version 4.0c for Macintosh (GraphPad Software).
      Protein Crystallization—Crystals of apo-PPARγ (
      • Ebdrup S.
      • Pettersson I.
      • Rasmussen H.B.
      • Deussen H.J.
      • Frost Jensen A.
      • Mortensen S.B.
      • Fleckner J.
      • Pridal L.
      • Nygaard L.
      • Sauerberg P.
      ) were obtained by vapor diffusion at 20 °C using sitting drop made by mixing 2 μl of protein solution (6 mg/ml, in 20 mm Tris, 5 mm DTT, 0.5 mm EDTA, pH 8.0) with 1 μl of reservoir solution (0.8 m sodium citrate, 0.15 m Tris, pH 8.0). Prismatic crystals (200 × 200 × 200 μm) appeared after a few days. The crystals were soaked for 2-30 days in a storage solution (1 m sodium citrate, 0.15 m Tris, pH 8.0) containing the ligand (1 mm). Ligands dissolved in ethanol were diluted in the storage solution so that the final ethanol concentration was 1%. Crystals were flash-cooled in liquid nitrogen after a fast soaking in a cryoprotectant buffer (storage solution with glycerol 20% (v/v)). Crystals belong to the space group C2 with cell parameters a = 93.14 Å, b = 60.95 Å, c = 118.11 Å, β = 103.3° for the complex with the R-enantiomer and a = 93.54 Å, b = 60.91 Å, c = 118.35 Å, β = 103.1° for that with the S-enantiomer (crystal soaked 30 days). The asymmetric unit is formed by one homodimer (53% solvent).
      Structure Determination—X-ray data were collected at 100 K under a nitrogen stream using synchrotron radiation (beamline XRD1 at Elettra, Trieste). The diffracted intensities were processed using the programs DENZO and SCALEPACK (
      • Otwinowski Z.
      • Minor W.
      ). Refinement was performed with CNS (
      • Brunger A.T.
      • Adams P.D.
      • Clore G.M.
      • DeLano W.L.
      • Gros P.
      • Grosse-Kunstleve R.W.
      • Jiang J.S.
      • Kuszewski J.
      • Nilges M.
      • Pannu N.S.
      • Read R.J.
      • Rice L.M.
      • Simonson T.
      • Warren G.L.
      ) using the coordinates of apo-PPARγ (
      • Nolte R.T.
      • Wisely G.B.
      • Westin S.
      • Cobb J.E.
      • Lambert M.H.
      • Kurokawa R.
      • Rosenfeld M.G.
      • Willson T.M.
      • Glass C.K.
      • Milburn M.V.
      ) (Protein Data Bank code 1PRG) as a starting model. All data between 8 and 2.1 Å (8-2.25 Å for the S-enantiomer) were included with σ cut-off of 2.0. The statistics of crystallographic data and refinement are summarized in Table 1.
      TABLE 1Statistics of crystallographic data and refinement
      (R)-1(S)-1
      Structure from crystals soaked 30 days.
      (S)-1
      Structure from crystals soaked 6 h.
      Wavelength (Å)1.01.01.0
      Temperature (K)100100100
      Cell dimensions (Å)93.14; 60.95; 118.1193.54; 60.91; 118.3593.00;60.14;118.08
      β angle (degrees)103.26103.08103.07
      Resolution range (Å)30.0-2.10 (2.17-2.10)
      The values in parentheses refer to the outer shell.
      30.0-2.10 (2.18-2.10)
      The values in parentheses refer to the outer shell.
      30.0-2.25 (2.33-2.25)
      The values in parentheses refer to the outer shell.
      Space groupC2C2C2
      No. of unique reflections32,49136,70629,624
      Rsym (%)4.04.05.2
      I/σ(I)24.2 (3.1)
      The values in parentheses refer to the outer shell.
      25.8 (2.1)
      The values in parentheses refer to the outer shell.
      16.9 (2.9)
      The values in parentheses refer to the outer shell.
      Completeness (%)85.9 (77.8)
      The values in parentheses refer to the outer shell.
      96.3 (92.7)
      The values in parentheses refer to the outer shell.
      97.7 (94.7)
      The values in parentheses refer to the outer shell.
      R factor (%)24.727.223.8
      Rfree27.829.529.0
      r.m.s. deviation bonds (Å)0.0090.0080.007
      r.m.s. deviation angles (degrees)1.3351.6191.185
      No. of water mol.187116198
      No. of protein atoms2165
      The value refers to one monomer.
      2165
      The value refers to one monomer.
      2165
      The value refers to one monomer.
      No. of ligand atoms33
      The value refers to one monomer.
      33
      The value refers to one monomer.
      33
      The value refers to one monomer.
      a Structure from crystals soaked 30 days.
      b Structure from crystals soaked 6 h.
      c The values in parentheses refer to the outer shell.
      d The value refers to one monomer.

      RESULTS

      (R)-1 and (S)-1 Activate PPARγ-mediated Transcription—The activity of the two enantiomers of a novel compound deriving from the ureidofibrate class (Fig. 1) was evaluated on a panel of nuclear receptors in a Gal4-based assay. The results in Fig. 2A show that (R)-1 and (S)-1 only activate the transcription of PPARα and PPARγ, whereas the activity of the other nuclear receptors, including the PPARβ/δ subtype, is not affected by these two molecules.
      Figure thumbnail gr2
      FIGURE 2(R)-1 and (S)-1 selectively activate PPARα and γ. The specificity of (S)-1 (5 μm) and (R)-1 (1 μm) was assessed in a co-transfection assay in HepG2 cells using expression vectors for Gal4-nuclear receptor-LBD fusion proteins as indicated in A. ctrl, samples treated with vehicle (0.1% ethanol). Shown are concentration-response curves of rosiglitazone, (S)-1, and (R)-1 in HepG2 cells co-transfected with p5xGal4UAS reporter and pGal4-hPPARγ-LBD (B) and in co-activator recruitment assay (C). D, antagonism of (S)-1 against rosiglitazone (1 and 10 μm) in a co-activator recruitment assay. Ki of ligands was determined by displacing [3H]rosiglitazone with increasing concentrations of cold ligands in a scintillation proximity assay (E).
      To gain more insights on the potency and efficacy of these new compounds on the PPARγ subtype, we carried out transfection assays with the human Gal4-PPARγ-LBD expression vector and compared the curves obtained with increasing concentrations of (R)-1 and (S)-1 with the corresponding data obtained with rosiglitazone, here used as a reference compound. As shown in Fig. 2B,(R)-1 is able to activate PPARγ, but its potency is clearly lower than that of rosiglitazone (Table 2). (S)-1 is less potent than the R-enantiomer toward PPARγ, and in addition its efficacy is markedly lower than that of rosiglitazone and (R)-1 (Table 2). Co-activator recruitment assays (Fig. 2C) are consistent with the results of cellular assays and show that (S)-1 is less potent and has lower efficacy than (R)-1 and rosiglitazone (Table 2). The determination of binding constants by a scintillation proximity assay confirms that (S)-1 is a lower affinity PPARγ ligand, whereas the affinity of (R)-1 is comparable with that of rosiglitazone (Fig. 2E).
      TABLE 2Potency and efficacy of tested ligands toward PPARγ as determined in Gal4-based assays (top) and in cell-free co-activator recruitment assays (bottom)
      Tested ligandEC50Efficacy
      nm%
      Rosiglitazone3.8 ± 1.5100.0 ± 10.5
      (R)-173.3 ± 49.9116.1 ± 9.0
      (S)-1593.0 ± 114.150.4 ± 5.1
      Tested ligandKdΔmax
      nm%
      Rosiglitazone14.8 ± 6.6100.0 ± 4.9
      (R)-1684.8 ± 106.870.5 ± 2.5
      (S)-11,978 ± 523.523.3 ± 3.2
      (S)-1 Behaves Like a PPARγ Partial Agonist—Since the efficacy of (S)-1 in cell-based assays is 50%, and that in the co-activator recruitment assay is about 25% of that of rosiglitazone (Table 2), we wanted to test whether this molecule could behave as a partial agonist of PPARγ. The co-activator recruitment assay (Fig. 2D) shows that when 1 or 10 μm rosiglitazone is incubated with increasing concentrations of (S)-1, the percentage of efficacy decreases to about 25%, a value corresponding to the maximal efficacy reached by (S)-1. These results suggest that (S)-1 is a partial agonist of PPARγ.
      Overall Structure of the LBD—Having observed the different behavior of the two enantiomers, we solved the crystal structure of PPARγ complexed with (R)-1 and (S)-1. The structure of PPARγ-LBD in both complexes is very similar to that of the apo-form, with an r.m.s. deviation between Cα atoms of 0.31Å (225 Cα pairs). The r.m.s. deviation between the structures of the two complexes is only 0.20 Å (267 Cα pairs), the largest deviation regarding the terminal part of helix 12 (Cα 473-476). The LBD domain consists of 12 helices and a small β-sheet of four strands. The electron density corresponding to residues 260-275, belonging to the so-called omega loop, is badly defined or missing. The residues of this region were not considered in the refinement. A homodimer forms the asymmetric unit in both structures. The two monomers, denoted as A and B, contain one ligand molecule. Helix 12 of monomer A is in its active conformation. As observed in other PPAR structures, helix 12 in monomer B does not adopt the active conformation, despite the presence of the ligand (
      • Cronet P.
      • Petersen J.F.
      • Folmer R.
      • Blomberg N.
      • Sjoblom K.
      • Karlsson U.
      • Lindstedt E.L.
      • Bamberg K.
      ). This is because H12 of monomer B is engaged in several contacts with a symmetry-related molecule of monomer A. Particularly, it occupies the hydrophobic groove into which the coactivator peptide binds (nuclear receptor-box) (
      • Nolte R.T.
      • Wisely G.B.
      • Westin S.
      • Cobb J.E.
      • Lambert M.H.
      • Kurokawa R.
      • Rosenfeld M.G.
      • Willson T.M.
      • Glass C.K.
      • Milburn M.V.
      ,
      • Shiau A.K.
      • Barstad D.
      • Radek J.T.
      • Meyers M.J.
      • Nettles K.W.
      • Katzenellenbogen B.S.
      • Katzenellenbogen J.A.
      • Agard D.A.
      • Greene G.L.
      ,
      • Darimont B.D.
      • Wagner R.L.
      • Apriletti J.W.
      • Stallcup M.R.
      • Kushner P.J.
      • Baxter J.D.
      • Fletterick R.J.
      • Yamamoto K.R.
      ), thus mimicking the two-turn amphipathic coactivator α helix (supplemental Fig. 2).
      Binding of the R-Enantiomer in the PPARγ-LBD—The electron density reported in Fig. 3A clearly shows the position of the R-enantiomer. Its molecular volume (∼435 Å3) fills about 30% of the large T-shaped cavity volume (∼1440 Å3) limited by the H3, H5, H7, H12, and the β-sheet. The carboxylate group of the ligand participates in a hydrogen bonding network (Fig. 4A). One of the carboxylate oxygens forms a hydrogen bond of 2.6 Å with the His323 Nϵ2 atom. The other carboxylate oxygen is engaged in a bifurcated hydrogen bond of 2.4 and 2.6 Å, respectively, with the Tyr473 hydroxyl oxygen and His449 Nϵ2 atom. An additional hydrogen bond (2.8 Å) is formed by the ether oxygen of the ligand with the His449 Nζ2 atom.
      Figure thumbnail gr3
      FIGURE 3A, 2Fo - Fc electron density map calculated around the R-enantiomer (shown in yellow) and contoured at 1σ; B, 2Fo - Fc electron density map calculated around the S-enantiomer (shown in cyan) and contoured at 0.9σ.
      Figure thumbnail gr4
      FIGURE 4A, hydrogen bonding network between the R-enantiomer (yellow) and the LBD of PPARγ (the triad His323, His449, and Tyr473 is colored in orange); B, hydrogen bonding network between the S-enantiomer (cyan) and the LBD of PPARγ (the triad and Ser289 are in purple).
      The methyl and ethyl substituents of the asymmetric carbon of the ligand form several hydrophobic interactions with Leu residues in the loop 11/12 and in H11 and H12 helices (Fig. 5A), further stabilizing the active conformation of H12. The ligand methyl group is 3.5 Å away from one of the methyls of Leu469, whereas the methyl of the ethyl group forms contacts of 3.9, 3.5, 3.4, and 3.7 Å, respectively, with the Leu469, Leu465, and Leu453 methyl groups.
      Figure thumbnail gr5
      FIGURE 5A, hydrophobic interactions of the R-enantiomer (yellow) with Leu residues (white) of LBD; B, hydrophobic interactions of the S-enantiomer (cyan) with Leu residues (white) of LBD.
      The long aliphatic chain and the fused ring system of the R-enantiomer, respectively, occupy the lower and upper part of the distal cavity, there making hydrophobic contacts with the surrounding protein residues. In the region of the distal cavity, the final Fourier difference map shows residual electron density that was attributed to several interlinked water molecules, suggesting that this region could still accommodate additional substituents. In fact, the PPAR ligand binding pocket is larger than that of other nuclear receptors, explaining its capability to accept a variety of naturally occurring and synthetic lipophilic acids.
      Mutagenesis of the Leu465, Leu469, and Ile472 Residues of PPARγ—The importance of these residues of the C-terminal portion of PPARγ in the activation mediated by (R)-1 was studied by mutagenesis analysis, which shows that the basal activity of these mutants is severely impaired (Fig. 6). Neither rosiglitazone nor (R)-1 could reach activity levels comparable with those of the activated wild type receptor (Fig. 6).
      Figure thumbnail gr6
      FIGURE 6Activation of PPARγ point mutants. Shown is the effect of rosiglitazone and (R)-1 on the transcriptional activity of PPARγ L465A, L469A, and I472A mutants as measured by the induction of luciferase reporter activity on wild type (wt) and point mutant PPARγ-GAL4 chimeras. Data are expressed as mean ± S.D. of relative reporter activity with the corresponding -fold activation as compared with vehicle indicated above the bars. Both compounds were tested at 1 μm. N/D, the EC50 was not determined because the receptor was not activated by the ligand.
      Binding of the S-Enantiomer in the PPARγ-LBD—The electron density of the S-enantiomer in the PPARγ ligand cavity (Fig. 3B) is interrupted at the central part of the molecule, and it is generally less well defined than that of its enantiomer. The location of the polar head is about 1 Å away from the triad His323, His449, and Tyr473 and from the helix 12, as compared with that of the R-enantiomer (Fig. 7). However, the fused ring systems of the two ligands, almost perpendicular to each other, and their long aliphatic chains maintain unshifted positions in the cavity because of different torsion angles involving the central aromatic ring and the hexocyclic nitrogen atom. The 1-Å shift adopted by the S-enantiomer is probably required to reduce the steric clash between its ethyl group and the Gln286 backbone belonging to helix 3 (Fig. 7). Such ligand displacement gives rise to a different, noncanonical, hydrogen bonding network of its polar head. As can be seen in Fig. 4B, one of the ligand carboxylate oxygens forms three hydrogen bonds of 2.4, 2.7, and 3.1 Å, respectively, with Tyr473 OH, His323, and His449 Nϵ2 atoms. The other carboxylate oxygen is engaged in a hydrogen bond of 2.4 Å with the Ser289 OH. Moreover, the hydrophobic interactions between the ligand and the Leu residues in the loop 11/12 and in H11 and H12 helices reduce to only one favorable contact of 4.0 Å with the Leu453 methyl and the methyl on the asymmetric carbon atom, as shown in Fig. 5B.
      Figure thumbnail gr7
      FIGURE 7Cα superposition of the complexes with the R- and the S-enantiomer (in yellow and cyan, respectively). Protein side chains of the complex with the R-enantiomer are shown in green; the correspondent side-chains are in pink for the complex with the S-enantiomer.
      The Fourier difference map clearly shows residual electron density in the ligand cavity that has been attributed to a water molecule. This water forms a hydrogen bond with the Tyr473 OH, faces the positively charged His449 and negatively charged ligand polar head on one side, and, on the other side, faces a hydrophobic wall formed by the Leu side chains belonging to the loop 11/12 and the helix 11. There, this water forms repulsive contacts of 3.1 and 2.8 Å, respectively, with the methyl group of the ligand and of Leu453 (Fig. 7).
      A comparison between the two complexes reveals that, although the His323 and His449 residues maintain the same orientation, a rearrangement for the backbone atoms of the critical residue of H12, Tyr473, occurs in the complex with the S-enantiomer, so that this helix is slightly shifted from its active conformation (Fig. 7). Fig. 8A shows the orientation of the Tyr473 side chain that is supported by the polarization interactions between the aromatic π-cloud and the surrounding methyl groups of Val450, Leu453, Leu476, and Ile472, occurring in the complex with the R-enantiomer. Fig. 8B shows the same view for the complex with the S-enantiomer. In this case, the Tyr473 side chain interacts only with the Leu453 methyl group, which causes an evident destabilization of H12, also denoted by the increase of the B values (20-30%) of residues 472-474 of the complex with the S-enantiomer.
      Figure thumbnail gr8
      FIGURE 8A, hydrophobic contacts between Tyr473 (green) and nonpolar residues (white) of the protein complex with the R-enantiomer (yellow). B, hydrophobic contacts between Tyr473 (green) belonging to H12 and apolar residues (white) of the protein complex with the S-enantiomer (cyan).
      Another Crystal Structure of the PPARγ·S-Enantiomer Complex—The structure of the complex between PPARγ and the S-enantiomer, obtained from crystals of the apo-form soaked for only6hinthe ligand solution, was also determined and compared with that previously reported, resulting from crystals soaked for more than 30 days. Intriguingly, the comparison of the two structures reveals significant differences in the electron density for the ligand and several protein residues and gives additional insights into the conformational changes induced by accommodating the ligand in the LBD. It is likely, therefore, that we have observed, by freezing the crystal at different soaking times, two different thermodynamic states of the ligand bound into the cavity. The hypothesis of equilibrium of multiple coexisting conformations of nuclear receptors in the presence of a partial agonist needs to be supported by further molecular dynamics simulations. However, the ligand binding comparison between the R- and the S-enantiomer, previously described, has been made, choosing the structure of the complex with the S-enantiomer showing a suboptimal, probably inactive, conformation of H12, which belongs to the crystal soaked for 1 month.
      In the structure of crystal soaked 6 h, the S-enantiomer carboxylate group participates in a hydrogen bonding network with the His323, His449, and Tyr473 almost identical to that found for the R-enantiomer. However, the ligand ethyl substituent gives rise to repulsive interactions with the Gln286 backbone of helix 3 (Fig. 9). In fact, missing and weak electron density at the expected positions of the ethyl group and Gln286 side chain, respectively, have been observed (supplemental Fig. 3A). Modeling the ethyl group gives rise to repulsive interactions in all cases. Moreover, the electron density around the Tyr473 side chain, positioned as in the complex with the R-enantiomer, is well defined (supplemental Fig. 4A), and the water molecule, here hydrogen-bonded to one of the ligand carboxylate oxygens, forms short contacts with one of the methyls of Leu453 (2.8 Å) and of Leu465 (3.3 Å). At variance with this situation, the observed displacement of about 1 Å found in the complex obtained after ∼30 days of soaking reduces the steric clash between the ligand ethyl group and the Gln286 residue, as evidenced by well defined electron density maps in the regions of the above groups (supplemental Fig. 3B). Simultaneously, the Tyr473 side chain is rotated as shown in Fig. 9, and the water molecule approaching the Leu465 residue forces its side chain to a different conformation. This conformational change is transmitted, through the Met463 side chain, to the Phe282 aromatic ring, resulting in a general rearrangement of the loop 11/12 (supplemental Fig. 5).
      Figure thumbnail gr9
      FIGURE 9Cα superposition of the two crystal structures with the S-enantiomer, obtained after different times of soaking. The ligand and the contacting protein residues are in green in the 6-h soaking crystal structure and in purple in the 30-day soaking crystal structure.
      Mutagenesis of the Gln286 Residue of PPARγ—To validate the functional importance of the steric clash between the ethyl group of (S)-1 and Gln286, we attempted to eliminate it by substituting Gln286 with a glycine. However, this substitution reduces dramatically the basal activity and prevents the activation of the receptor by (S)-1 (Fig. 10A). Conversely, rosiglitazone activates the mutated receptor to levels comparable with the wild type although only at micromolar concentration (Fig. 10A). Since the Q286G mutant did not allow us to demonstrate the functional relevance of this residue in the steric clash, we synthesized the achiral compound 2 bearing the shorter methyl group in place of the ethyl group (Fig. 1). As shown in Fig. 10B, compound 2 is more potent than (S)-1, but the efficacy is lower than that of rosiglitazone. This result strongly argues for the presence of the steric clash between the ethyl group of (S)-1 and Gln286.
      Figure thumbnail gr10
      FIGURE 10A, the effect of rosiglitazone and (S)-1 on the transcriptional activity of PPARγ Q286G mutant as measured by the induction of luciferase reporter activity on wild type (wt) and point mutant PPARγ-GAL4 chimera. Data are expressed as relative reporter activity with the corresponding -fold activation as compared with vehicle indicated above the bars. Both compounds were tested at 1 μm. All data are expressed as mean ± S.D. B, concentration-response curves of rosiglitazone and compound 2 on Gal4-PPARγ-LBD assessed in cotransfection assays in HepG2 cells as described in the legend to .

      DISCUSSION

      We have identified two ureidofibrate-like enantiomers that display different behavior toward PPARγ. The R-enantiomer is a more potent agonist than the corresponding S-enantiomer, which has the typical behavior of a PPARγ partial agonist, since its efficacy is lower than that of rosiglitazone and the R-enantiomer, and it decreases the coactivator recruitment on PPARγ induced by rosiglitazone. It should be noted that the differences of the values obtained in the cell-based reporter gene assay and in the in vitro co-activator recruitment assay (Table 2) may be explained by the fact that the recruitment assay measures the binding of a peptide containing a single nuclear receptor box to the LBD of PPARγ. Therefore, the recruitment of ligands in this type of assay may underestimate the ability of ligand-induced co-activator recruitment to induce transcription in a cell-based reporter gene assay. In addition, as opposed to the cell-free co-activator recruitment assay, in the cellular environment more co-activators are present at the same time, and they may cooperate in activating gene transcription.
      Structural Basis for the Mechanism of Partial Agonism—The transmission of information through structural changes is fundamental to the function of many proteins. It is well known that in nuclear receptors, the dynamic behavior of helix 12 plays a key role in coactivator recruitment. Ligand binding to the LBD modulates the stabilization of H12 in different ways; it functions as a trigger that acts directly, stabilizing its active conformation (
      • Love J.D.
      • Gooch J.T.
      • Benko S.
      • Li C.
      • Nagy L.
      • Chatterjee V.K.
      • Evans R.M.
      • Schwabe J.W.
      ), or indirectly, influencing the H12 stability through additional structural changes, such as repositioning of H11 or different conformations of the loop 11/12. H12 stability is also promoted by the action of the ligand as LBD global stabilizer (
      • Cronet P.
      • Petersen J.F.
      • Folmer R.
      • Blomberg N.
      • Sjoblom K.
      • Karlsson U.
      • Lindstedt E.L.
      • Bamberg K.
      ,
      • Pissios P.
      • Tzameli I.
      • Kushner P.
      • Moore D.D.
      ).
      Full agonists shift the equilibrium between active and inactive conformation of helix 12 toward the active state, leading to coactivator recruitment. Partial agonists are incapable of shifting this equilibrium in favor of the active conformation to the same extent as full agonists; they are not able to stabilize the H12 in the proper position, due to the lack of a few key interactions, and even a slight mispositioning of H12 may result in an attenuated transcriptional response (
      • Renaud J.P.
      • Moras D.
      ,
      • Steinmetz A.C.
      • Renaud J.P.
      • Moras D.
      ).
      Previous work on estrogen receptors showed that a high affinity ligand can induce a low affinity coactivator binding site, behaving as a partial agonist, by selecting for suboptimal conformation of H11 that, in turn, destabilizes the agonist position of H12 through loss of hydrophobic or electrostatic contacts (
      • Nettles K.W.
      • Sun J.
      • Radek J.T.
      • Sheng S.
      • Rodriguez A.L.
      • Katzenellenbogen J.A.
      • Katzenellenbogen B.S.
      • Greene G.L.
      ).
      Moreover, in a structural study on the human mineralocorticoid receptor, it has been proposed that the residues of the loop 11/12 might be compared with a zip fastener, in which each residue of the loop 11/12 is a link, which helps to promote the precise positioning of H12 (
      • Hellal-Levy C.
      • Fagart J.
      • Souque A.
      • Wurtz J.M.
      • Moras D.
      • Rafestin-Oblin M.E.
      ).
      Differences in the hydrophobic packing of this loop may contribute to different H12 dynamics (
      • Nettles K.W.
      • Sun J.
      • Radek J.T.
      • Sheng S.
      • Rodriguez A.L.
      • Katzenellenbogen J.A.
      • Katzenellenbogen B.S.
      • Greene G.L.
      ). However, to date there is still little structural information to explain the reduced efficacy observed with high affinity partial agonist ligands (such as genistein for estrogen receptor β) (
      • Nettles K.W.
      • Sun J.
      • Radek J.T.
      • Sheng S.
      • Rodriguez A.L.
      • Katzenellenbogen J.A.
      • Katzenellenbogen B.S.
      • Greene G.L.
      ,
      • Pike A.C.
      • Brzozowski A.M.
      • Hubbard R.E.
      • Bonn T.
      • Thorsell A.G.
      • Engstrom O.
      • Ljunggren J.
      • Gustafsson J.A.
      • Carlquist M.
      ).
      To gain further insights at the molecular level into the behavior of a ligand as a partial agonist, we solved the structures of PPARγ complexes with the two enantiomeric forms of a ureidofibrate-like derivative, both active toward PPARγ, the R-enantiomer behaving as a full agonist and the S-enantiomer as a partial agonist (Table 2, Fig. 2). These model compounds are particularly suitable to study the mechanism of partial agonism, because they differ only for the switching of a methyl with an ethyl group on the asymmetric carbon atom, a small structural change that causes a significant difference in the pharmacological profile.
      Although helix 12 is in a similar conformation in both complexes, there are some important differences. In the crystal complex PPARγ·(R)-1, the active conformation of H12 is stabilized by the following interactions: (a) both carboxylate oxygens of the ligand engage canonical hydrogen bonds with the three residues His323, His449, and Tyr473 involved in the receptor activation (Fig. 4A); (b) the appropriate position of the Tyr473 aromatic side chain is ensured by polarization interactions with Ile472 and Leu476 on one side and with Val450 and Leu453 on the other side (Fig. 8A); (c) the ligand methyl and ethyl groups form several favorable hydrophobic interactions with Leu453 of H11, Leu469 of H12, and Leu465 of the loop 11/12 (Fig. 5A). Thus, the potency of the R-enantiomer is a direct consequence of a very effective stabilization of the helix 12, through hydrophobic and electrostatic interactions. Moreover, helix 12 is here stabilized in the proper conformation to recruit the coactivator, the same observed in other crystal structures of complexes with full agonists (
      • Cronet P.
      • Petersen J.F.
      • Folmer R.
      • Blomberg N.
      • Sjoblom K.
      • Karlsson U.
      • Lindstedt E.L.
      • Bamberg K.
      ,
      • Ebdrup S.
      • Pettersson I.
      • Rasmussen H.B.
      • Deussen H.J.
      • Frost Jensen A.
      • Mortensen S.B.
      • Fleckner J.
      • Pridal L.
      • Nygaard L.
      • Sauerberg P.
      ,
      • Xu H.E.
      • Lambert M.H.
      • Montana V.G.
      • Plunket K.D.
      • Moore L.B.
      • Collins J.L.
      • Oplinger J.A.
      • Kliewer S.A.
      • Gampe Jr., R.T.
      • McKee D.D.
      • Moore J.T.
      • Willson T.M.
      ,
      • Sauerberg P.
      • Pettersson I.
      • Jeppesen L.
      • Bury P.S.
      • Mogensen J.P.
      • Wassermann K.
      • Brand C.L.
      • Sturis J.
      • Woldike H.F.
      • Fleckner J.
      • Andersen A.S.
      • Mortensen S.B.
      • Svensson L.A.
      • Rasmussen H.B.
      • Lehmann S.V.
      • Polivka Z.
      • Sindelar K.
      • Panajotova V.
      • Ynddal L.
      • Wulff E.M.
      ,
      • Gampe Jr., R.T.
      • Montana V.G.
      • Lambert M.H.
      • Miller A.B.
      • Bledsoe R.K.
      • Milburn M.V.
      • Kliewer S.A.
      • Willson T.M.
      • Xu H.E.
      ).
      In the complex with the S-enantiomer (crystals soaked for 30 days), a 1-Å shift of the ligand away from helix 12 is observed. This is probably caused by a steric clash between the ligand ethyl group and the Gln286 backbone. Even if the H12 conformation only slightly differs from that observed in the complex with the R-enantiomer, its stability appears completely different for the following aspects: (a) only one of the carboxylate oxygens of the ligand engages hydrogen bonds with the three residues His323, His449, and Tyr473 (Fig. 4B); (b) the 1-Å shift of the ligand reduces favorable hydrophobic contacts with helix 12 to only one (Fig. 5B); (c) a water molecule, situated between the ligand and hydrophobic residues of the loop 11/12, prevents further productive ligand-receptor binding interactions (Fig. 9); (d) the Tyr473 aromatic ring adopts a different orientation, forming van der Waals interactions only with Leu453 of H11 (Fig. 8B).
      Effect of L465A and L469A Mutations on the Potency of (R)-1—As expected, the shortening of the side chain at positions 465 and 469 shows a reduction of activity of (R)-1 due to the lack of the important hydrophobic contacts between the methyl and ethyl substituents of the ligand and the methyl groups of the leucines (Fig. 5A). However, the strong reduction of activity observed with the mutated aporeceptor could also be ascribed to the role played by these two leucines in stabilizing H12 through direct contacts with H3. In fact, they form favorable contacts with the side chain of the key residue Gln286, belonging to H3. Leu465 also interacts with the ring of Tyr473, stabilizing the active conformation of this residue.
      Effect of I472A on the Potency of (R)-1—Also, in this case there is a dramatic reduction of the potency of (R)-1 toward the I472A mutant. This could be determined by the loosening of the contact with Tyr473 and by the lack of a short hydrophobic contact made by a methyl of Ile472 with a carbon atom of the H323 ring (3.4 Å). This contact helps to stabilize the correct orientation of this very important residue, involved in hydrogen bonds with the Tyr473 ring and the carboxylate oxygens of the ligand. This hydrophobic contact is not present in the structure with the (S)-1 ligand (4.4 Å), where the Ile472 and His323 side chains slightly changed their orientations.
      Effect of the Q286G Mutation on the Potency of (S)-1Fig. 11 is a superposition among the crystal complexes formed by PPARγ with rosiglitazone, (R)-1, and (S)-1, respectively. There, the relative positions adopted by the three ligands in the binding cavity can be deduced. In the complex with rosiglitazone, the Gln286 NH2 group forms a hydrogen bond with the ligand CO, and it is at 3.1 Å from one of the Leu465 methyls, working as a bridge between the ligand and the loop 11/12 preceding helix 12. If the Gln286 side chain maintained the same position in the complexes with (R)-1 and (S)-1, the methyl of (R)-1 and the ethyl of (S)-1 would form short contacts with the Gln286 Cγ atom (2.9 and 1.8 Å, respectively). To release these repulsive interactions, a reorientation of the Gln286 side chain takes place in both complexes. In the new position that the Gln286 side chain adopts in the complexes with (R)-1 and (S)-1, its NH2 forms a hydrogen bond of 3.2 and 3.0 Å, respectively, with the carbonyl oxygen of Ser464 belonging to loop 11/12. Moreover, it is at 3.1 and 3.0 Å from one of the Leu465 methyls. From these observations, it is evident that in the three complexes, the Gln286 residue plays a key role in stabilizing helix 12, and the Q286G mutation has a dramatic effect on the receptor activity. Because it is not possible to confirm the functional relevance of the steric clash between Gln286 and the ethyl group of (S)-1 by mutating this residue, we tested a ligand with the shorter methyl group replacing the ethyl group. As expected, this substitution improves the potency of this ligand as compared with that of (S)-1, thus confirming that this steric clash does play a role in determining the behavior of the S-enantiomer.
      Figure thumbnail gr11
      FIGURE 11Superposition of the Cα traces of the complexes of PPARγ with the R-enantiomer (green), the S-enantiomer (cyan), and rosiglitazone (purple). Putative contacts are shown in parentheses.
      Molecular Adaptation Caused by the Entry of the Partial Agonist—Two crystal structures with the S-enantiomer, collected after different soaking times, offer two snapshots depicting different nuclear receptor conformations provoked by the binding of a partial agonist in the LBD. The comparison of the two structures shows that in one of these (6 h of soaking), the helix 12 is in the canonical transcriptionally active conformation, whereas in the other, H12 shows a suboptimal, probably inactive conformation. In the structure from crystals soaked for 6 h, a steric hindrance between the ethyl group of the ligand and the backbone of H3 (Gln286) is present. In the structure from crystals soaked for 1 month, the ligand, in the attempt to reduce the steric hindrance with Gln286, displaces its position, weakening the hydrogen bond with Tyr473. This residue, consequently, changes its position; a water molecule, bound to Tyr473, shields a zone of hydrophobic interactions between ligand and Leu465 of the loop 11/12, forcing the Leu465 side chain to interact with that of Met463. This, in turn, changes its position, influencing that of Phe282 of H3. The relevance of a bulky, properly oriented, hydrophobic residue at this site in stabilizing the loop, and indirectly H12, was highlighted in a recent work on RAR, where a mutation at the corresponding position (W225A in RAR) created a constitutive repressor that failed to bind co-activator (
      • Benko S.
      • Love J.D.
      • Beladi M.
      • Schwabe J.W.
      • Nagy L.
      ). PPARγ Phe282 occupies a key position to form van der Waals contacts with several residues in the loop 11/12 and in H11 (supplemental Fig. 5).
      The final result of this general rearrangement of the LBD leads to a loss of hydrophobic interaction between the ligand and the apolar residues of H12, H11, and loop 11/12, destabilizing helix 12, as shown by the lack of well defined density of its crucial residue Tyr473 (supplemental Fig. 4, A and B). This residue is now oriented in a less productive way, losing the important hydrophobic interaction with Val450 (Figs. 8B and 12). The importance of this site is well highlighted in the work on RAR discussed above (
      • Benko S.
      • Love J.D.
      • Beladi M.
      • Schwabe J.W.
      • Nagy L.
      ), where a mutation at this position (A392R in RAR) led to a constitutively active receptor. In that case, the larger Arg side chain, with respect to that of Ala, could form several new interactions with helices 4 and 12, stabilizing the active conformation of H12.
      Figure thumbnail gr12
      FIGURE 12Superposition of the Cα traces of the complexes of PPARγ with the R-enantiomer (yellow), the S-enantiomer (green), and GW0072 (purple). Tyr473, Leu453, and Val450 are drawn as sticks.
      Even in the crystal structure of PPARγ complexed with the weak partial agonist GW0072 (
      • Oberfield J.L.
      • Collins J.L.
      • Holmes C.P.
      • Goreham D.M.
      • Cooper J.P.
      • Cobb J.E.
      • Lenhard J.M.
      • Hull-Ryde E.A.
      • Mohr C.P.
      • Blanchard S.G.
      • Parks D.J.
      • Moore L.B.
      • Lehmann J.M.
      • Plunket K.
      • Miller A.B.
      • Milburn M.V.
      • Kliewer S.A.
      • Willson T.M.
      ), which does not interact at all with helix 12, a similar orientation of the Tyr473 aromatic ring, lacking the important contact with Val450, has been observed. Fig. 12 represents a superposition of the receptor backbones adopted in the crystal complexes with our R- and S-enantiomers and with GW0072. There, a progressive reorientation of the Tyr473 side chain can be noted. Its aromatic ring is stabilized by polarization interactions with both Val450 and Leu453 residues in the complex with the almost full agonist R-enantiomer, whereas it faces only the Leu453 residue in the complex with the partial agonist S-enantiomer and the weak partial agonist GW0072. In the complex with GW0072, there is also an evident displacement of helix 11, which further destabilizes H12. The three crystal complexes can represent different states of stability of the helix 12, with the PPARγ·GW0072 clearly representing the less stable one. A similar behavior has been observed for the structures of the complexes with the other known PPARγ partial agonists (
      • Ostberg T.
      • Svensson S.
      • Selen G.
      • Uppenberg J.
      • Thor M.
      • Sundbom M.
      • Sydow-Backman M.
      • Gustavsson A.L.
      • Jendeberg L.
      ,
      • Burgermeister E.
      • Schnoebelen A.
      • Flament A.
      • Benz J.
      • Stihle M.
      • Gsell B.
      • Rufer A.
      • Ruf A.
      • Kuhn B.
      • Marki H.P.
      • Mizrahi J.
      • Sebokova E.
      • Niesor E.
      • Meyer M.
      ,
      • Lu I.L.
      • Huang C.F.
      • Peng Y.H.
      • Lin Y.T.
      • Hsieh H.P.
      • Chen C.T.
      • Lien T.W.
      • Lee H.J.
      • Mahindroo N.
      • Prakash E.
      • Yueh A.
      • Chen H.Y.
      • Goparaju C.M.
      • Chen X.
      • Liao C.C.
      • Chao Y.S.
      • Hsu J.T.
      • Wu S.Y.
      ).
      Conclusions—In the present work, we argue that the partial agonist behavior of the S-enantiomer could be ascribed to a destabilization of the active conformation of helix 12. A suboptimal conformation of this helix was observed in one of the two structures of the complex with the same partial agonist, suggesting the coexistence in solution of transcriptionally active and inactive forms and probably explaining the dramatic lack of efficacy in co-activator recruitment and in transactivation activity.
      Finally, it will be interesting to test whether the PPARγ partial agonist (S)-1 can recruit a different set of co-activators. This feature, along with the capacity to activate also the PPARα subtype, may aid the identification of Selective PPARγ modulators and will help the design of new agents characterized by improved pharmacological properties and at the same time reduced side effects typical of other known PPARγ agonists.

      Acknowledgments

      We thank Drs. John Schwabe (Medical Research Council, Cambridge, UK) and Enrique Saez (Genomics Institute of the Novartis Research Foundation, San Diego, CA) for critically reading the manuscript and for helpful discussion. We are grateful to Drs. Iannis Talianidis, Krister Bamberg for kindly providing the pGal4-hepatocyte nuclear factor 4, pGal4-PPARα and -γ, pHis-PPARγ, and p5xGal4UAS vectors and to Elda Desiderio Pinto for administrative assistance.

      Supplementary Material

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