FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2117639596> ?p ?o ?g. }

• W2117639596 endingPage "38828" @default.
• W2117639596 startingPage "38821" @default.
• W2117639596 abstract "The structures of the liver X receptor LXRβ (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 Å, respectively. Together with its isoform LXRα (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRβ structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed “agonist conformation” of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor. The structures of the liver X receptor LXRβ (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 Å, respectively. Together with its isoform LXRα (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRβ structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed “agonist conformation” of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor. Liver X receptors (LXR) 1The abbreviations used are: LXR, liver X receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; FXR, farnesoid X receptor; LBD, ligand-binding domain; ABCA1, gene encoding ATP binding cassette protein A1; ABCG1, gene encoding ATP binding cassette protein G1; AF-2, activation function 2; EPPS, 4-(2-hydroxy-ethyl)-1-piperazinepropanesulfonic acid.1The abbreviations used are: LXR, liver X receptor; RXR, retinoid X receptor; PPAR, peroxisome proliferator-activated receptor; FXR, farnesoid X receptor; LBD, ligand-binding domain; ABCA1, gene encoding ATP binding cassette protein A1; ABCG1, gene encoding ATP binding cassette protein G1; AF-2, activation function 2; EPPS, 4-(2-hydroxy-ethyl)-1-piperazinepropanesulfonic acid. are members of the superfamily of nuclear receptors. These transcription factors regulate target genes through a dynamic series of interactions with specific DNA response elements as well as transcriptional coregulators. The binding of ligand has profound effects on these interactions and has the potential to trigger both gene activation and, in some cases, gene silencing. There are 48 sequence-related nuclear receptors in humans and the family comprises receptors that recognize hormones, both steroidal and non-steroidal, but also receptors responding to metabolic intermediates and to xenobiotics. There are also a number of so-called orphan receptors where the natural ligand is unknown. Some of the receptors show a very specific and high affinity ligand binding, like the thyroid hormone receptors, whereas others have a substantially lower affinity for their ligands and are less discriminating in their ligand selectivity. Like many of the other non-steroid hormone receptors, LXR functions as a heterodimer with the retinoid X receptor (RXR) to regulate gene expression (1Willy P.J. Mangelsdorf D.J. Genes Dev. 1997; 11: 289-298Crossref PubMed Scopus (141) Google Scholar, 2Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). Together with peroxisome proliferator-activated receptor (PPAR) and farnesoid X receptor (FXR), LXRs represent a subclass of so-called permissive RXR heterodimers. In this subclass, the RXR heterodimers can be activated independently by either the RXR ligand, the partner's ligand, or synergistically by both (3Peet D.J. Janowski B.A. Mangelsdorf D.J. Curr. Opin. Genet. Dev. 1998; 8: 571-575Crossref PubMed Scopus (320) Google Scholar).LXRs consist of two closely related receptor isoforms encoded by separate genes, LXRα (NR1H3) and LXRβ (NR1H2). LXRα shows tissue-restricted expression with the highest mRNA levels in the liver and somewhat lower levels in the kidney, small intestine, spleen, and adrenal gland (4Apfel R. Benbrook D. Lernhardt E. Ortiz M.A. Salbert G. Pfahl M. Mol. Cell Biol. 1994; 14: 7025-7035Crossref PubMed Scopus (291) Google Scholar, 5Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (908) Google Scholar). In contrast, LXRβ is ubiquitously expressed (6Teboul M. Enmark E. Li Q. Wikstrom A.C. Pelto-Huikko M. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2096-2100Crossref PubMed Scopus (198) Google Scholar, 7Song C. Kokontis J.M. Hiipakka R.A. Liao S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10809-10813Crossref PubMed Scopus (208) Google Scholar). Both LXR isoforms can be activated by specific oxysterols that are formed in vivo (2Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar, 8Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1441) Google Scholar, 9Janowski B.A. Grogan M.J. Jones S.A. Wisely G.B. Kliewer S.A. Corey E.J. Mangelsdorf D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 266-271Crossref PubMed Scopus (780) Google Scholar). In view of the high degree of homology between the LXR isoforms (75% identity in the ligand-binding domain (LBD), 54% identity overall), it is perhaps not surprising that few subtype-specific biological responses have been described and that information on subtype selective ligands is limited. LXRs have been shown to regulate several genes involved in cholesterol and lipid homeostasis. Prominent examples are the phospholipid/cholesteryl ester transporters ABCA1 (10Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (845) Google Scholar, 11Repa J.J. Turley S.D. Lobaccaro J.A. Medina J. Li L. Lustig K. Shan B. Heyman R.A. Dietschy J.M. Mangelsdorf D.J. Science. 2000; 289: 1524-1529Crossref PubMed Scopus (1144) Google Scholar, 12Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar, 13Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (831) Google Scholar) and ABCG1 (14Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) and the sterol response element-binding protein (SREBP1c) (15Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1401) Google Scholar) that induces fatty acid synthesizing enzymes. Increasing insight into the involvement of LXRs in cholesterol and fatty acid homeostasis has led to considerable interest in LXRs from the pharmaceutical point of view. As an example, one hallmark of atherosclerosis is the buildup of cholesteryl esters in macrophages of the arterial wall, transforming the cells into foam cells that are constituents of the atherosclerotic plaque. The potential to increase cholesterol efflux from macrophages/foam cells by inducing genes such as ABCA1 and/or G1, thereby preventing or even reversing the atherosclerotic process, makes LXRs highly interesting drug targets. This notion is supported by recent reports pointing to the importance of LXRs as inhibitors of foam cell formation and thereby also of atherosclerosis (16Schuster G.U. Parini P. Wang L. Alberti S. Steffensen K.R. Hansson G.K. Angelin B. Gustafsson J.-Å. Circulation. 2002; 106: 1147-1153Crossref PubMed Scopus (156) Google Scholar, 17Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. Wang X. Lusis A.J. Tontonoz P. Schulman I.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11896-11901Crossref PubMed Scopus (370) Google Scholar).Our understanding of how nuclear receptor ligands exert their effects has been dramatically enhanced by the elucidation of the crystal structures of nuclear receptors either in their apo-state or with an agonist or antagonist bound to the LBD (18Wagner R.L. Apriletti J.W. Mcgrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (805) Google Scholar, 19Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1052) Google Scholar, 20Bourguet W. Vivat V. Wurtz J.M. Chambon P. Gronemeyer H. Moras D. Mol. Cell. 2000; 5: 289-298Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 21Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.-Å. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2917) Google Scholar, 22Gampe 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. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 23Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1022) Google Scholar, 24Williams S.P. Sigler P.B. Nature. 1998; 393: 392-396Crossref PubMed Scopus (575) Google Scholar, 25Xu H.E. Stanley T.B. Montana V.G. Lambert M.H. Shearer B.G. Cobb J.E. McKee D.D. Galardi C.M. Plunket K.D. Nolte R.T. Parks D.J. Moore J.T. Kliewer S.A. Willson T.M. Stimmel J.B. Nature. 2002; 415: 813-817Crossref PubMed Scopus (509) Google Scholar). The LBDs share a common, mainly α-helical, fold that embeds a hydrophobic ligand-binding pocket. The structures have revealed that ligands can affect the conformation of the ligand-dependent activation function 2 residing in the C-terminal H12. An agonist allows H12 to cover the ligand-binding pocket, thereby providing one of the sides in the coactivator-binding pocket. Conversely, an antagonist induces an H12 conformation blocking the coactivator-binding site and/or leading to recruitment of corepressors (21Brzozowski A.M. Pike A.C. Dauter Z. Hubbard R.E. Bonn T. Engstrom O. Ohman L. Greene G.L. Gustafsson J.-Å. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2917) Google Scholar, 25Xu H.E. Stanley T.B. Montana V.G. Lambert M.H. Shearer B.G. Cobb J.E. McKee D.D. Galardi C.M. Plunket K.D. Nolte R.T. Parks D.J. Moore J.T. Kliewer S.A. Willson T.M. Stimmel J.B. Nature. 2002; 415: 813-817Crossref PubMed Scopus (509) Google Scholar, 26Nolte 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. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1660) Google Scholar). The binding modes of several of these coregulators have been described in detail (26Nolte 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. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1660) Google Scholar, 27Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Genes Dev. 1998; 12: 3343-3356Crossref PubMed Scopus (824) Google Scholar, 28Shiau A.K. Barstad D. Loria P.M. Cheng L. Kushner P.J. Agard D.A. Greene G.L. Cell. 1998; 95: 927-937Abstract Full Text Full Text PDF PubMed Scopus (2224) Google Scholar).Given its biological and pharmaceutical importance in cholesterol homeostasis, it is of great interest to determine the three-dimensional structure of LXR LBD. Synthetic, high-affinity ligands have great utility in this respect. Here we report structures of LXRβ in complex with two very different synthetic agonist ligands, T0901317 (29Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1380) Google Scholar) and GW3965 (30Collins J.L. Fivush A.M. Watson M.A. Galardi C.M. Lewis M.C. Moore L.B. Parks D.J. Wilson J.G. Tippin T.K. Binz J.G. Plunket K.D. Morgan D.G. Beaudet E.J. Whitney K.D. Kliewer S.A. Willson T.M. J. Med. Chem. 2002; 45: 1963-1966Crossref PubMed Scopus (367) Google Scholar), respectively.EXPERIMENTAL PROCEDURESLXRβ Protein Preparation—Human LXRβ-LBD (Gly-213-Glu-461 (NH1H2)) N-terminal-tagged with MGSSHHHHHHSSGLVPRGSHM was overexpressed in Escherichia coli BL21 Star™ (DE3) cells (Invitrogen), using the pET28a expression system. Fermentation was carried out in batch culture (2× Luria Bertani medium, 22 °C), and expression of the recombinant protein was induced by the addition of 0.55 mm isopropyl-β-d-thiogalactoside at A600 = 5.0. After the 4-h induction, the cells were harvested by centrifugation. The cell pellet was resuspended and washed once with buffer (20 mm HEPES, pH 8.0, 100 mm KCl, 10% glycerol, and 2.5 mm monothioglycerol). Final cell pellet was frozen at -70 °C.40-g bacteria cells were lysed by glass beadbeater (BioSpec Products, Inc.) in extract buffer containing 50 mm TRIS, pH 8.8, 250 mm NaCl, 10% glycerol, and 1 mm phenylmethylsulfonyl fluoride. Soluble protein extract was collected by centrifugation at 11,000 rpm, 20 min in Sorvall RC-5B centrifuge (DuPont instrument AB), GSA rotor. Crude LXRβ was eluted from 25 ml of Talon by 20 mm TRIS, pH 8.0, 100 mm imidazole. Further purification was performed by anion-exchange chromatography (5 ml of Hitrap Q FF, Amersham Biosciences), and LXRβ was eluted by applying a gradient from 0 to 250 mm NaCl, pH 8.0. After thrombin cleavage, the final LXRβ (6-7 mg) fraction was obtained by running 4% polyacrylamide native gel electrophoresis in TRIS-EPPS buffer system.Crystallization and Data Collection—Crystallization was carried out using the hanging drop vapor diffusion technique. Both LXRβ-T0901317 and LXRβ-GW9365 crystals were grown from buffer containing 8.5% isopropanol, 17% polyethylene glycol 4000, 85 mm HEPES, pH 7.5, and 15% glycerol at room temperature.The first LXRβ-T0901317 crystals formed in the P6122 space group, with a = b = 58.7, c = 293.8 and diffracted to better than 3 Å. In the same drops another crystal form was later detected belonging to the P212121 space group. Before data collection, the crystals were flash-frozen in the 100-K nitrogen gas stream of an Oxford cryostream700. Data were either collected with an MAR345 image plate detector using X-rays from a Rigaku H3R rotating anode generator + Osmic Confocal Max-Flux™ optics or with an ADSC Q4R CCD at Experimental Station ID14-4 at European Synchrotron Radiation Facility (ESRF). The observed reflections were reduced, merged, and scaled with MOSFLM (31Leslie A.G.W. Joint CCP4 and ESF-EAMBC Newsletter on Protein Crystallography. 1992; 26Google Scholar) and Scala (32Evans P.R. Joint CCP4 and ESF-EAMBC Newsletter on Protein Crystallography. 1997; 33: 22-24Google Scholar) in the CCP4 package (33Collaborative Computational Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). Details of data collection are summarized in Table I.Table IData collection and refinement statisticsLXRβ-T0901317 Split ligandLXRβ-T0901317 Unsplit ligandLXRβ-GW3965Data collectionSourceID14 EH4 ESRFRigaku RuH3RID14 EH4 ESRFSpace groupP212121P212121P212121Unit cell parameters:a58.958.758.7b103.6103.398.9c176.4176.0175.8Resolution (last shell)2.1 Å (2.1-2.21)2.8 Å (2.8-2.95Å)2.4 (2.4-2.53)ObservationsUnique622812715337733Total23198392460129438Completeness (%) (last shell)99.8 (100.0)99.9 (99.7)98.5 (95.4)<I>/<σ(I)> (last shell)5.0 (2.7)7.6 (1.9)8.8 (3.5)R sym (%) (last shell)aRsym=∑hkl∑|I(h,k,l,i)-〈I(h,k,l)〉|∑hkl∑I(h,k,l,i) .6.6 (27.6)8.4 (40.2)5.0 (21.8)Refinement (last shell)2.1Å (2.1-2.155)2.8 (2.8-2.872)2.4 (2.4-2.462)R work (%) (last shell)bR=∑hkl∥Fobs(h,k,l)|-k|Fcalc(h,k,l)∥∑hkl|Fobs(h,k,l)| .24.3 (25.3)19.5 (27.9)20.7 (21.8)R free (%) (last shell)bR=∑hkl∥Fobs(h,k,l)|-k|Fcalc(h,k,l)∥∑hkl|Fobs(h,k,l)| .27.9 (29.3)26.2 (34.8)26.3 (29.6)Number of atoms777977827673R.m.s.d. bonds (Å)cR.m.s.d., root mean square deviation.0.0150.0160.016R.m.s.d. angles (°)cR.m.s.d., root mean square deviation.1.381.491.36Average B-factor (Å2)20.224.323.1R work is calculated from a set of reflections in which 5% of the total reflections have been randomly omitted from the refinement and used to calculate R free.a Rsym=∑hkl∑|I(h,k,l,i)-〈I(h,k,l)〉|∑hkl∑I(h,k,l,i) .b R=∑hkl∥Fobs(h,k,l)|-k|Fcalc(h,k,l)∥∑hkl|Fobs(h,k,l)| .c R.m.s.d., root mean square deviation. Open table in a new tab Structure Determination and Refinement—The structure was determined by molecular replacement methods with the CCP4 version of AMoRe (34Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar), using an LXRβ homology model based on a thyroid hormone receptor β structure. 2M. Farnegardh, unpublished result. The molecular replacement was done on the first 3 Å data of LXRβ-T0901317 crystallized in P6122 and revealed one monomer per asymmetric unit. The crystal packing along one of the two folds revealed that the protein formed a tight homodimer, which allowed us to use the homodimer to search the second crystal form P212121 that gave two homodimers in the asymmetric unit. Electron densities for the T0901317 ligand confirmed the solutions of the molecular replacement. Model building was done with O (35Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar) and refined with Refmac (33Collaborative Computational Project 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar, 36Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1648) Google Scholar) and manual rebuilding in O. The four monomer complexes were treated as single TLS groups in Refmac, which gave more interpretable electron density maps and improved the R-factors substantially. Statistics of the data collection and refinement are summarized in Table I.The Final Model—The region between H1 and H3 (residues 243-259), part of the β-sheet between H6 and H7 (residues 329-332), and the loop connecting H11 and H2 (residues 445-447) have been abolished or modeled as alanines in some of the protein complexes because of weak electron densities. H12 (residues 439-460) is absent in molecule C of the GW3965-complexed protein. The geometry of the final model is good, and only one amino acid residue, Leu-330, has been found to be an outlier in the Ramachandran plot. In some of the structures where Leu-330 has an ordered conformation it has an energetically unfavorable conformation.Computation—Protein cavity volumes were calculated using the VOIDOO program from the Uppsala Software Factory package (47Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (976) Google Scholar), using the Connolly molecular surface representation. Figs. 1, 2, and 6 were generated using Molscript (37Kraulis P. J. App. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and Raster3D (38Merrit E. Murphy M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar). Fig. 3, A-C, and Fig. 4, A and B, were generated using PyMOL (39DeLano, W. L. (2002) DeLano Scientific, San Carlos, CAGoogle Scholar).Fig. 2Overall comparison of the LXR complexes to PPARγ Stereo image showing Cα trace of protein and bonds of the LXRβ-T0901317 complex in green, the LXRβ-GW3965 complex in yellow, the LXRβ apoform (blue), and for comparison the PPARγ-rosiglitazone complex in red. Every 20th residue in LXRβ has been marked.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Stereo view of the binding pockets of LXRβ bound to T0901317 (carbon atoms colored black), GW3965 (carbon atoms colored green (protein) and yellow (ligand)), and the apoform (carbon atoms colored blue), respectively. The structures were superimposed using LSQMAN (46Kleywegt G.J. Jones T.A. Methods Enzymol. 1997; 277: 525-545Crossref PubMed Scopus (303) Google Scholar) (0.7 Å root mean square deviation over 219 Cα atoms using a 3.8 Å cutoff) The carbon atoms and hydrogen bonds of the residues in the T0901317 and the GW3965 LXRβ complexes were colored gray and green, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3F obs-F calc electron densities (the blue mesh) at the ligand-binding sites contoured at 3.0 σ The electron densities were calculated after omitting the ligand. A, T0901317 complex of synchrotron-collected complex reveals that the ligand is cleaved by the x-rays. B, T0901317 complex of the laboratory source rapid data collection where no cleavage of the ligand has occurred. C, GW3965 complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The cavities (yellow mesh) of the ligand-binding sites of LXRβ-T0901317 (A) and LXRβ-GW3965 (B) complexes, respectively, located between H12 and the β-sheet. Cavities described under “Results” and “Discussion” are marked around the binding cavity. The loop following H1, N-terminal part of H3 and H7 was positioned above the binding cavity and has been removed in this view for clarity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSThe Overall Structure of LXRβ—Attempts to crystallize LXRα and LXRβ with or without coactivator peptides containing an LXXLL motif were performed. LXRβ without peptide was the first to produce sufficiently good crystals for structural analysis. The structures of the LXRβ-T0901317 and LXRβ-GW3965 complexes were determined by molecular replacement and refined, including all diffraction data, to 2.1 and 2.4 Å, respectively. Four LXRβ complexes form the asymmetric unit with the chain names A, B, C, and D, respectively. Ligand occupies all four LBD molecules in the respective complexes with the exception of molecules C in the GW3965 co-crystallized protein, which appears to lack most of its ligand. 185 and 167 defined water molecules, respectively, were also included in the refinement.The nomenclature of the secondary structure elements is based on that of the thyroid hormone receptor (18Wagner R.L. Apriletti J.W. Mcgrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (805) Google Scholar) and has been applied throughout this report even when comparing to other nuclear receptors where the original authors used a different nomenclature. The overall structure of the LXRβ LBD encompasses residues 220-460. Regions with weak electron densities were not built (for details see “Experimental Procedures”).Overall, the structure comprises a core layer of three helices (H5/6, H9 and H10) sandwiched between two additional layers of helices (H1-4 and H7, H8, and H11, respectively) and represents a typical nuclear receptor LBD fold (Figs. 1 and 2). This arrangement creates a wedge-shaped molecular scaffold that contains a wider upper part, which shows the highest degree of sequence conservation between different nuclear receptor LBDs. The lower, narrower, part is folded to form a hydrophobic cavity into which the ligand can bind. The remaining secondary elements, an antiparallel β-sheet comprising three strands and H12 (sometimes referred to as the AF-2 motif), reside on either side of the ligand-binding cavity. The crystal structures of the LXRβ complexes contain a homodimer of the protein with H10 and H11 of the two monomer LBDs forming the dimer interface related by a C2 symmetry axis (Fig. 1). The dimer interface is consistent with the ones found for other homodimer structures such as PPARγ (26Nolte 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. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1660) Google Scholar) and RXRα (19Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1052) Google Scholar) and also with the heterodimer structure of RXRα/RARα (20Bourguet W. Vivat V. Wurtz J.M. Chambon P. Gronemeyer H. Moras D. Mol. Cell. 2000; 5: 289-298Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), which also has the same C2 symmetry axis between the monomers. In the PPARγ/RXRα structure a dimer with the same secondary elements was observed, but the dimer interface is asymmetric where the PPAR monomer is rotated about 10° from the C2 symmetry axis of the RXR monomer (22Gampe 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. Mol. Cell. 2000; 5: 545-555Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). It was postulated that this asymmetry together with an interaction of PPARγ H12 and H8 and H11 of RXR would be the explanation for the permissiveness of PPARγ and that this would apply to the permissive LXR and FXR receptors as well.T0901317 Is Cleaved by X-rays—The ligand-binding site revealed a clear density corresponding quite well to the T0901317 ligand. The ligand could easily be fitted with its hexafluoroisopropanol aniline group close to H12, and the results gave no doubts about the orientation of the ligand. The trifluoroethyl group could also be fitted in a nice density, but the sulfophenyl group did not appear to be connected to the rest of the molecule. There was a clear gap in the electron density between the nitrogen and sulfur atoms (Fig. 3A). A more detailed analysis on a rotating anode source revealed that it is the x-rays that split the ligand. 2M. Farnegardh, unpublished result. The consequence of the cleavage is that the sulfophenyl group moves away from the rest of the ligand and the distance between the nitrogen and sulfur atom increases by 1 Å from what would be anticipated in an intact ligand. A data set was collected to 2.8 Å on a rotating anode for only 12 h to get data with no detectable cleavage of the ligand (Fig. 3B). These data were used to build a model of the intact ligand in the binding site and are used under the remaining “Results” and “Discussion.” The shift in the position of the sulfophenyl group is the only detectable difference between structures obtained with cleaved and non-cleaved ligand.Structure of the T0901317-LXRβ Complex—The entire ligand-binding pocket extends from H12 to the β-sheet lying between helices H6 and H7. Its volume varies between the four protein complexes mainly depending on the position of the β-hairpin loop residue Phe-329 and Arg-319. These residues are fairly disordered and show different positional preferences in the four molecules. Depending on their positions, the volume of the boot-shaped cavity ranges from 560 to 680 Å3 (Fig. 4A). The ankle shaft is buried in a cavity (C1) formed by H12 and H11 on one side and H7 and H8 on the other side. Residues from H8, H7, and H6 form the heel of the boot, (cavity 2 (C2)), while the toes are covered by the β-sheet and H3, forming cavity 3 (C3). In its entirety, the pocket is about 12.5 Å in length along the boot shaft down to the heel (C1-C2) and 8 Å from the heel to the toes (C2-C3). The thickness of the boot shaft down to the toes is ∼6.5 Å.Ligand recognition is achieved through a combination of one specific hydrogen bond and the complementarity of the binding cavity to the non-polar character of the T0901317 ligand (Figs. 4A, 5A, and 6). The hexafluoroisopropanol-substituted aniline group occupies C1, and the acidic hydroxyl oxygen atom is in good position to donate a proton to the Nϵ imidazole nitrogen of His-435. The imidazole ring is firmly positioned by a group of three water molecules stretching out to the surface between H12 and the bend between H5 and H6. Water molecule W1 is tetrahedrally coordinated by the imidazole Nδ, Ser-436, the carbonyl oxygen of residue 432, and a water molecule, W2, at the surface. The bend releases the carbonyl oxygen of residue 305 and the nitrogen of residue 309 to coordinate another water molecule, W3, that is further tetrahedrally coordinated by Trp-457 Nϵ and water molecule W2. Otherwise, Phe-268, Thr-272, Leu-345, Gln-438, Val-439, Leu-442, Leu-449, Leu-453, and Trp-457 surround the hexafluoroisopropanol group. The ligand continues down via the aniline through these residues to the aniline nitrogen atom. The trifluoroethyl group of the nitrogen nicely enters into the C2 cavity and is completely embedded by Leu-31" @default.
• W2117639596 created "2016-06-24" @default.
• W2117639596 creator A5013991412 @default.
• W2117639596 creator A5018917040 @default.
• W2117639596 creator A5019924767 @default.
• W2117639596 creator A5024467012 @default.
• W2117639596 creator A5036193907 @default.
• W2117639596 creator A5049469017 @default.
• W2117639596 creator A5069164271 @default.
• W2117639596 creator A5079753696 @default.
• W2117639596 date "2003-10-01" @default.
• W2117639596 modified "2023-10-11" @default.
• W2117639596 title "The Three-dimensional Structure of the Liver X Receptor β Reveals a Flexible Ligand-binding Pocket That Can Accommodate Fundamentally Different Ligands" @default.
• W2117639596 cites W1570388980 @default.
• W2117639596 cites W1744821831 @default.
• W2117639596 cites W1907321242 @default.
• W2117639596 cites W1975070287 @default.
• W2117639596 cites W1980161952 @default.
• W2117639596 cites W1980222251 @default.
• W2117639596 cites W1981873124 @default.
• W2117639596 cites W1996127793 @default.
• W2117639596 cites W1996168740 @default.
• W2117639596 cites W2000577172 @default.
• W2117639596 cites W2001641653 @default.
• W2117639596 cites W2003840075 @default.
• W2117639596 cites W2013083986 @default.
• W2117639596 cites W2013378362 @default.
• W2117639596 cites W2022546192 @default.
• W2117639596 cites W2024851295 @default.
• W2117639596 cites W2028231353 @default.
• W2117639596 cites W2029354866 @default.
• W2117639596 cites W2039664177 @default.
• W2117639596 cites W2042901562 @default.
• W2117639596 cites W2046658442 @default.
• W2117639596 cites W2048397414 @default.
• W2117639596 cites W2061357909 @default.
• W2117639596 cites W2078248419 @default.
• W2117639596 cites W2080476827 @default.
• W2117639596 cites W2081732968 @default.
• W2117639596 cites W2085909854 @default.
• W2117639596 cites W2086317930 @default.
• W2117639596 cites W2088436733 @default.
• W2117639596 cites W2092190477 @default.
• W2117639596 cites W2093296161 @default.
• W2117639596 cites W2101738855 @default.
• W2117639596 cites W2103794058 @default.
• W2117639596 cites W2104606103 @default.
• W2117639596 cites W2111650618 @default.
• W2117639596 cites W2112200653 @default.
• W2117639596 cites W2112632255 @default.
• W2117639596 cites W2113456624 @default.
• W2117639596 cites W2135007868 @default.
• W2117639596 cites W2141662437 @default.
• W2117639596 cites W2147596285 @default.
• W2117639596 cites W2153673003 @default.
• W2117639596 cites W2165104851 @default.
• W2117639596 doi "https://doi.org/10.1074/jbc.m304842200" @default.
• W2117639596 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12819202" @default.
• W2117639596 hasPublicationYear "2003" @default.
• W2117639596 type Work @default.
• W2117639596 sameAs 2117639596 @default.
• W2117639596 citedByCount "148" @default.
• W2117639596 countsByYear W21176395962012 @default.
• W2117639596 countsByYear W21176395962013 @default.
• W2117639596 countsByYear W21176395962014 @default.
• W2117639596 countsByYear W21176395962015 @default.
• W2117639596 countsByYear W21176395962016 @default.
• W2117639596 countsByYear W21176395962017 @default.
• W2117639596 countsByYear W21176395962018 @default.
• W2117639596 countsByYear W21176395962019 @default.
• W2117639596 countsByYear W21176395962020 @default.
• W2117639596 countsByYear W21176395962021 @default.
• W2117639596 countsByYear W21176395962022 @default.
• W2117639596 countsByYear W21176395962023 @default.
• W2117639596 crossrefType "journal-article" @default.
• W2117639596 hasAuthorship W2117639596A5013991412 @default.
• W2117639596 hasAuthorship W2117639596A5018917040 @default.
• W2117639596 hasAuthorship W2117639596A5019924767 @default.
• W2117639596 hasAuthorship W2117639596A5024467012 @default.
• W2117639596 hasAuthorship W2117639596A5036193907 @default.
• W2117639596 hasAuthorship W2117639596A5049469017 @default.
• W2117639596 hasAuthorship W2117639596A5069164271 @default.
• W2117639596 hasAuthorship W2117639596A5079753696 @default.
• W2117639596 hasBestOaLocation W21176395961 @default.
• W2117639596 hasConcept C107824862 @default.
• W2117639596 hasConcept C116569031 @default.
• W2117639596 hasConcept C12554922 @default.
• W2117639596 hasConcept C170493617 @default.
• W2117639596 hasConcept C185592680 @default.
• W2117639596 hasConcept C55493867 @default.
• W2117639596 hasConcept C8010536 @default.
• W2117639596 hasConcept C86803240 @default.
• W2117639596 hasConceptScore W2117639596C107824862 @default.
• W2117639596 hasConceptScore W2117639596C116569031 @default.
• W2117639596 hasConceptScore W2117639596C12554922 @default.
• W2117639596 hasConceptScore W2117639596C170493617 @default.
• W2117639596 hasConceptScore W2117639596C185592680 @default.
• W2117639596 hasConceptScore W2117639596C55493867 @default.

• next