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• W2894803492 abstract "Nuclear receptor farnesoid X receptor (FXR) functions as the major bile acid sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. Because of its central role in metabolism, FXR represents an important drug target to manage metabolic and other diseases, such as primary biliary cirrhosis and nonalcoholic steatohepatitis. FXR and nuclear receptor retinoid X receptor α (RXRα) form a heterodimer that controls the expression of numerous downstream genes. To date, the structural basis and functional consequences of the FXR/RXR heterodimer interaction have remained unclear. Herein, we present the crystal structures of the heterodimeric complex formed between the ligand-binding domains of human FXR and RXRα. We show that both FXR and RXR bind to the transcriptional coregulator steroid receptor coactivator 1 with higher affinity when they are part of the heterodimer complex than when they are in their respective monomeric states. Furthermore, structural comparisons of the FXR/RXRα heterodimers and the FXR monomers bound with different ligands indicated that both heterodimerization and ligand binding induce conformational changes in the C terminus of helix 11 in FXR that affect the stability of the coactivator binding surface and the coactivator binding in FXR. In summary, our findings shed light on the allosteric signal transduction in the FXR/RXR heterodimer, which may be utilized for future drug development targeting FXR. Nuclear receptor farnesoid X receptor (FXR) functions as the major bile acid sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. Because of its central role in metabolism, FXR represents an important drug target to manage metabolic and other diseases, such as primary biliary cirrhosis and nonalcoholic steatohepatitis. FXR and nuclear receptor retinoid X receptor α (RXRα) form a heterodimer that controls the expression of numerous downstream genes. To date, the structural basis and functional consequences of the FXR/RXR heterodimer interaction have remained unclear. Herein, we present the crystal structures of the heterodimeric complex formed between the ligand-binding domains of human FXR and RXRα. We show that both FXR and RXR bind to the transcriptional coregulator steroid receptor coactivator 1 with higher affinity when they are part of the heterodimer complex than when they are in their respective monomeric states. Furthermore, structural comparisons of the FXR/RXRα heterodimers and the FXR monomers bound with different ligands indicated that both heterodimerization and ligand binding induce conformational changes in the C terminus of helix 11 in FXR that affect the stability of the coactivator binding surface and the coactivator binding in FXR. In summary, our findings shed light on the allosteric signal transduction in the FXR/RXR heterodimer, which may be utilized for future drug development targeting FXR. The farnesoid X receptor (FXR, NR1H4) 2The abbreviations used are: FXRfarnesoid X receptorRXRretinoid X receptorNRnuclear receptorPPARperoxisome proliferator activator receptorTRthyroid hormone receptorRARretinoic acid receptorLBDligand-binding domainAF2activation function-29cRA9-cis-retinoic acidFXREFXR response elementIR1single inverted repeatSCAstatistic coupling analysisSRC1steroid receptor coactivator 1PDBProtein Data BankCCcorrelation coefficient. is a member of the nuclear receptor (NR) superfamily that plays a key role in the regulation of bile acids, lipid, and glucose metabolisms, as well as anti-inflammatory response and hepatocarcinogenesis inhibition (1Lee F.Y. Lee H. Hubbert M.L. Edwards P.A. Zhang Y. FXR, a multipurpose nuclear receptor.Trends Biochem. Sci. 2006; 31 (16908160): 572-58010.1016/j.tibs.2006.08.002Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 2Wang Y.D. Chen W.D. Moore D.D. Huang W. FXR: a metabolic regulator and cell protector.Cell Res. 2008; 18 (18825165): 1087-109510.1038/cr.2008.289Crossref PubMed Scopus (261) Google Scholar). FXR, which is abundantly expressed in liver, kidney, and intestine, has been identified as the main bile acid sensor (3Wang H. Chen J. Hollister K. Sowers L.C. Forman B.M. Endogenous bile acids are ligands for the nuclear receptor FXR BAR.Mol. Cell. 1999; 3 (10360171): 543-55310.1016/S1097-2765(00)80348-210.1016/S1097-2765(00)80484-0Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). Over the years, FXR has been demonstrated to be an effective drug target to treat metabolic diseases, because FXR agonist obeticholic acid (OCA or INT747) has been successfully used in primary biliary cirrhosis and nonalcoholic steatohepatitis treatment (4Fiorucci S. Cipriani S. Mencarelli A. Baldelli F. Bifulco G. Zampella A. Farnesoid X receptor agonist for the treatment of liver and metabolic disorders: focus on 6-ethyl-CDCA.Mini. Rev. Med. Chem. 2011; 11 (21707532): 753-76210.2174/138955711796355258Crossref PubMed Scopus (68) Google Scholar). farnesoid X receptor retinoid X receptor nuclear receptor peroxisome proliferator activator receptor thyroid hormone receptor retinoic acid receptor ligand-binding domain activation function-2 9-cis-retinoic acid FXR response element single inverted repeat statistic coupling analysis steroid receptor coactivator 1 Protein Data Bank correlation coefficient. FXR belongs to the structurally conserved family of nuclear receptors that function as ligand-regulated transcription factors. It consists of an N-terminal DNA-binding domain, which targets the receptor to specific genes, coupled by a flexible linker to a ligand-binding domain (LBD). The LBD can bind small lipophilic ligands and then serves as the transcriptional switch. Like most NRs, regulation of transcription by FXR is a complex process that relies on the release of corepressors and the ligand-dependent recruitment of coactivator proteins to a surface on the LBD. This surface, usually formed by helices 3, 4, 5, and 12 in FXR, is also called the activation function-2 (AF2) surface. In turn, those coregulatory proteins mediate the interactions with the basal transcriptional machinery, resulting in repression or activation of transcription (5Darimont B.D. Wagner R.L. Apriletti J.W. Stallcup M.R. Kushner P.J. Baxter J.D. Fletterick R.J. Yamamoto K.R. Structure and specificity of nuclear receptor-coactivator interactions.Genes Dev. 1998; 12 (9808622): 3343-335610.1101/gad.12.21.3343Crossref PubMed Scopus (828) Google Scholar). FXR operates as a heterodimer with another nuclear receptor named retinoid X receptor-α (RXRα, NR2B1) in vivo (6Forman B.M. Goode E. Chen J. Oro A.E. Bradley D.J. Perlmann T. Noonan D.J. Burka L.T. Mcmorris T. Lamph W.W. Evans R.M. Weinberger C. Identification of a nuclear receptor that is activated by farnesol metabolites.Cell. 1995; 81 (7774010): 687-69310.1016/0092-8674(95)90530-8Abstract Full Text PDF PubMed Scopus (964) Google Scholar). RXRα appears to respond to endogenous ligands, such as 9-cis-retinoic acid (9cRA) (7Chambon P. A decade of molecular biology of retinoic acid receptors.FASEB J. 1996; 10 (8801176): 940-95410.1096/fasebj.10.9.8801176Crossref PubMed Scopus (2601) Google Scholar). FXR/RXR heterodimer binds to FXR response element (FXRE), which is mostly a single inverted repeat (IR1) in the promoter region of the target gene (8Schuetz E.G. Strom S. Yasuda K. Lecureur V. Assem M. Brimer C. Lamba J. Kim R.B. Ramachandran V. Komoroski B.J. Venkataramanan R. Cai H. Sinal C.J. Gonzalez F.J. Schuetz J.D. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome p450.J. Biol. Chem. 2001; 276 (11509573): 39411-3941810.1074/jbc.M106340200Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). RXRs also serve as obligate heterodimer partners for many of the subfamily 1 NRs, including receptors for peroxisome proliferator activators (PPARs), thyroid hormone (TR), retinoic acid (RAR), and vitamin D (9Mangelsdorf D.J. Evans R.M. The RXR heterodimers and orphan receptors.Cell. 1995; 83 (8521508): 841-85010.1016/0092-8674(95)90200-7Abstract Full Text PDF PubMed Scopus (2830) Google Scholar, 10Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.Nature. 1992; 358 (1324435): 771-77410.1038/358771a0Crossref PubMed Scopus (1520) Google Scholar). These different RXR heterodimers can be divided into three classes: permissive, conditional, and nonpermissive RXR heterodimers, based on their different response to RXR ligands (11Shulman A.I. Larson C. Mangelsdorf D.J. Ranganathan R. Structural determinants of allosteric ligand activation in RXR heterodimers.Cell. 2004; 116 (15016376): 417-42910.1016/S0092-8674(04)00119-9Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 12Forman B.M. Umesono K. Chen J. Evans R.M. Unique response pathways are established by allosteric interactions among nuclear hormone receptors.Cell. 1995; 81 (7758108): 541-55010.1016/0092-8674(95)90075-6Abstract Full Text PDF PubMed Scopus (567) Google Scholar). Generally, RXR heterodimers that contain PPAR, LXR, and FXR, which can be activated by ligands for either partner in the dimer, are permissive RXR heterodimers. Heterodimer RXR/RAR demonstrates conditional permissiveness, because full response to RXR ligands occurs only in the presence of an RAR agonist. The nonpermissive RXR heterodimers (e.g. RXR/TR, RXR/vitamin D receptor) usually do not respond to RXR ligands (12Forman B.M. Umesono K. Chen J. Evans R.M. Unique response pathways are established by allosteric interactions among nuclear hormone receptors.Cell. 1995; 81 (7758108): 541-55010.1016/0092-8674(95)90075-6Abstract Full Text PDF PubMed Scopus (567) Google Scholar). Dimerization renders the control of NR function intricate, because the signal across the heterodimer interface provides an exquisite degree of combinatorial control of transactivation (13Leblanc B.P. Stunnenberg H.G. 9-cis Retinoic acid signaling: changing partners causes some excitement.Genes Dev. 1995; 9 (7649469): 1811-181610.1101/gad.9.15.1811Crossref PubMed Scopus (132) Google Scholar). Within nuclear receptors, allostery is increasingly recognized as a common regulatory process initiated by DNA, ligands, and coregulators (14Fernandez E.J. Allosteric pathways in nuclear receptors: potential targets for drug design.Pharmacol. Ther. 2018; 183 (29080700): 152-15910.1016/j.pharmthera.2017.10.014Crossref PubMed Scopus (15) Google Scholar). Previous studies have defined an energetic coupled network of amino acids that mediates the allosteric regulation in RXR heterodimers using the statistic coupling analysis (SCA) method. It identifies a signal pathway within the heterodimer connecting the hydrophobic core of the AF2 surface from each receptor, but effects of the mutations in the network vary in different heterodimers (11Shulman A.I. Larson C. Mangelsdorf D.J. Ranganathan R. Structural determinants of allosteric ligand activation in RXR heterodimers.Cell. 2004; 116 (15016376): 417-42910.1016/S0092-8674(04)00119-9Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). More structural information is needed to delineate the SCA network at the atomic level. To date, a number of FXR–LBD crystal structures have been resolved in complex with a range of distinct ligands, which show that FXR possesses a highly flexible ligand-binding pocket. However, these structures only contained FXR–LBD; thus, it remained unclear whether the presence of RXRα would impact FXR’s structure and function. Further understanding of FXR regulation requires a more in-depth knowledge of the interactions between FXR and its binding partner RXRα. Herein, we present crystal structures of FXR–LBD complexed with coactivator and two novel FXR ligands and, for the first time, FXR/RXRα–LBD heterodimer complexes in the presence of novel FXR ligands and natural RXR ligand. In addition, we conducted in vitro studies to determine the transcriptional coactivator steroid receptor coactivator 1 (SRC1) binding affinities for the individual nuclear receptors and receptors in the heterodimer complex. Structure-guided mutagenesis experiments were further used to investigate the signaling between the heterodimers. The hFXR–LBD was crystallized in the presence of FXR agonists (HNC143 and HNC180) and SRC1 (fragment 741–761) with one LXXLL motif. The purified hFXR/RXRα–LBD heterodimer was crystallized in complex with FXR agonists (HNC143, HNC180, or GW4064), RXR natural ligand 9cRA, and a synthetic peptide derived from the coactivator SRC1–2 (fragment 685–700, the second LXXLL motif in SRC1) containing a single LXXLL motif. Purification of the coexpressed hFXR/RXRα–LBD heterodimer (see “Experimental procedures”) and addition of compounds and SRC1 peptide were critical for the complex crystallization. FXR–LBD/ligand structures were solved by the molecular-replacement method using previously determined FXR–LBD structure as the search probe. The heterodimer complex structures were also determined by molecular replacement method with the FXR and RXRα monomer structures as the searching models in two steps. Electron density maps showed clear features for the respective ligands in the structures, the LXXLL motifs of the SRC1 peptides, and each LBD, except for helix 2 in RXRα and some regions in FXR. The statistics for data collection and structure refinement are summarized in Table 1.Table 1Statistics of crystallographic data and refinementComplexHNC143–FXR–LBDHNC180–FXR–LBDHNC143–FXR/9cRA–RXRHNC180–FXR/9cRA–RXRGW4064–FXR/9cRA–RXRData collectionSpace groupP212121F23P41212P212121P43212Cell dimensionsa (Å)79.38160.3583.3889.53102.85b (Å)98.77160.3583.3896.13102.85c (Å)119.27160.35161.63114.24109.46α = β = γ (°)9090909090Resolution (Å)2.882.602.102.953.05Rpim0.086 (0.507)aThe values in parentheses refer to statistics in the highest resolution bin.0.032 (0.267)0.024 (0.247)0.110 (0.338)0.020 (0.274)I/σ6.9 (1.6)17.2 (3.3)18.9 (3.4)5.4 (2.0)19.4 (3.0)Completeness (%)99.9 (100)100 (100)100 (100)98 (98.8)99.8 (99.7)Redundancy6.2 (6.4)10.3 (10.4)13.1 (13.5)3.5 (3.6)13.8 (14.4)RefinementNo. of unique reflections20,74310,10732,44219,73711,002Rwork/Rfree (%)bRfactor = Σ|FP − FPcalc|/ΣFP, where FP and FPcalc are the observed and calculated structure factors, Rfree is calculated from a randomly chosen 5% of reflections that have never been used in refinement, and Rfactor is calculated for the remaining 95% of the reflections.21.1/26.219.6/25.119.8/24.622.1/28.222.5/28.8No. of atomsProteins39541982386073843638Ligand/ion76647612258Water41181171Average B-factorsProtein61.8684354.9126.3ligand53.50442.9937.149.5118.1RMSDcRMSD is the root-mean-square deviation from ideal geometry.Bond lengths (Å)0.01240.01280.0170.0090.0118Bond angles (°)1.59241.61421.7981.521.7318Ramachandran plotFavored (%)96.496.1996.6795.5794.01Allowed (%)2.122.973.333.525.07Outliers (%)1.480.8500.910.92a The values in parentheses refer to statistics in the highest resolution bin.b Rfactor = Σ|FP − FPcalc|/ΣFP, where FP and FPcalc are the observed and calculated structure factors, Rfree is calculated from a randomly chosen 5% of reflections that have never been used in refinement, and Rfactor is calculated for the remaining 95% of the reflections.c RMSD is the root-mean-square deviation from ideal geometry. Open table in a new tab The structures of the hFXR/RXRα LBD heterodimer complexes contain six components: two receptor LBDs, their respective ligands, and two SRC1 peptides (Fig. 1a). The heterodimer complex structure is similar to the previous published RXR LBD heterodimer complexes (15Gampe 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. Asymmetry in the PPARγ/RXRα crystal structure reveals the molecular basis of heterodimerization among nuclear receptors.Mol. Cell. 2000; 5 (10882139): 545-55510.1016/S1097-2765(00)80448-7Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar16Bourguet W. Vivat V. Wurtz J.M. Chambon P. Gronemeyer H. Moras D. Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains.Mol. Cell. 2000; 5 (10882070): 289-29810.1016/S1097-2765(00)80424-4Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 17Svensson S. Ostberg T. Jacobsson M. Norström C. Stefansson K. Hallén D. Johansson I.C. Zachrisson K. Ogg D. Jendeberg L. crystal structure of the heterodimeric complex of the LXRα and RXRβ ligand binding domains in a fully agonistic conformation.EMBO J. 2003; 22 (12970175): 4625-463310.1093/emboj/cdg456Crossref PubMed Scopus (235) Google Scholar, 18Suino K. Peng L. Reynolds R. Li Y. Cha J.Y. Repa J.J. Kliewer S.A. Xu H.E. The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization.Mol. Cell. 2004; 16 (15610733): 893-90510.1016/S1097-2765(04)00727-010.1016/j.molcel.2004.11.036Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 19Wallace B.D. Betts L. Talmage G. Pollet R.M. Holman N.S. Redinbo M.R. Structural and functional analysis of the human nuclear xenobiotic receptor PXR in complex with RXRα.J. Mol. Biol. 2013; 425 (23602807): 2561-257710.1016/j.jmb.2013.04.012Crossref PubMed Scopus (46) Google Scholar20Kojetin D.J. Matta-Camacho E. Hughes T.S. Srinivasan S. Nwachukwu J.C. Cavett V. Nowak J. Chalmers M.J. Marciano D.P. Kamenecka T.M. Shulman A.I. Rance M. Griffin P.R. Bruning J.B. Nettles K.W. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers.Nat. Commun. 2015; 6 (26289479)801310.1038/ncomms9013Crossref PubMed Scopus (88) Google Scholar). Both LBDs adopted the canonical three-layered α helical sandwich fold, with FXR containing 12 α-helices (H1 to H12) and RXR containing 10 α-helices and 2 β-strands. The FXR and RXRα ligands occupy their respective ligand-binding pockets, and both receptors adopt the active configuration, such that the H12 is folded against the main body of the LBD. This conformation generates a recognition surface (AF2 surface) constituted by mostly hydrophobic residues from H3, H4, and H12 of FXR and RXRα, which allow one SRC1 peptide to bind to each receptor. Two coactivator peptides fold as a two-turn α-helix in the heterodimer complex with the hydrophobic side chains (LXXLL) packed against the agonist-induced FXR and RXRα surfaces. HNC compounds (HNC143 and HNC180) are GW4064 analogues (Fig. 2, a–c), sharing the similar hydrophobic head that can bind in the same position in FXR ligand-binding pocket. Compared with GW4064 and HNC143, HNC180 has a more potent EC50 in a coactivator recruitment assay (Fig. 2h) and is much more active in cell transfection assays (Fig. 2i). Electron density maps allowed unambiguous placement of HNC143, HNC180, and GW4064 into the FXR ligand-binding pocket in the heterodimer structures (Fig. 2, d–f) and monomer structures (data not shown). Overall, these binding modes are similar to that adopted by GW4064 in the monomeric FXR–LBD (21Akwabi-Ameyaw A. Bass J.Y. Caldwell R.D. Caravella J.A. Chen L. Creech K.L. Deaton D.N. Jones S.A. Kaldor I. Liu Y. Madauss K.P. Marr H.B. McFadyen R.B. Miller A.B. Navas III, F. et al.Conformationally constrained farnesoid X receptor (FXR) agonists: naphthoic acid-based analogs of GW 4064.Bioorg. Med. Chem. Lett. 2008; 18 (18621523): 4339-434310.1016/j.bmcl.2008.06.073Crossref PubMed Scopus (104) Google Scholar). The nonsteroid ring system of HNC143 or HNC180 is sandwiched between two hydrophobic layers of residues mainly contributed from H3, H5, and H11. The large number of hydrophobic interactions observed between HNC compounds and FXR suggests that these interactions are important for the binding. Structure superposition of HNC143 and HNC180 with GW4064 in the heterodimer structures (Fig. 2g) shows that the tail end of compound HNC143 rotated significantly. This positional difference also exits in the FXR–LBD monomer structures (data not shown). In both FXR–LBD monomer and heterodimer structures with these three ligands, the carboxylate of HNC143 forms a hydrogen bond with His344, leading to stabilization of the loop between H5 and H6, whereas HNC180 and GW4064 interact with Arg331 (Fig. S2). For HNC180 and GW4064-bound structures, the segment between H5 and H6 in FXR–LBD forms a short helix in the monomer structures, whereas it is absent in the heterodimer structure because of a lack of electron density. These results suggest that FXR has considerable conformation dynamics upon binding with different ligands or forming heterodimer with RXR. Given the importance of heterodimerization in nuclear receptor signal pathway, we addressed the question whether association of FXR and RXRα modifies the intrinsic ability of the receptors to interact with coregulators. To this end, we purified FXR–LBD and RXRα–LBD with or without fused LXXLL motifs (see “Experimental procedures”) to examine whether nuclear receptor LBD dimerization can influence the coactivator binding. The fused SRC1 to one of the two receptors in the heterodimer was designed to assess the binding of biotin-labeled SRC1 to the nonfused receptor in coactivator binding assay. To validate this approach, FXR/RXR heterodimer fused with two SRC1 peptides was tested in the fluorescence anisotropy assay. The results show that there is no binding between the two-SRC1–fused heterodimer and fluorescein-labeled peptide (Fig. S1). We then examined the SRC1 binding affinity for FXR and RXR in the context of the heterodimer and in response to different ligands (Table S1). The affinity of SRC1676–700 LXXLL motifs binding to the FXR–LBD alone (Kd = 6.60 μm) is lower than binding to the FXR–LBD in the FXR/RXR–LBD heterodimer complex (Kd = 2.42 μm) (Fig. 3, a and c). A similar effect was found in RXRα–LBD (Fig. 3, b and d). Therefore, dimerization enhances the binding affinity of FXR–LBD and RXR–LBD for LXXLL motifs. In addition, contributing to the overall stability of the receptor, FXR agonists or RXR agonist (9cRA) can enhance the SRC1 binding, and the addition of 9cRA or FXR agonists will further increase the binding affinity in FXR/RXR·SRC1 or FXR·SRC1/RXR heterodimer (Fig. 3, e and f). We further tested the EC50 of FXR ligands in coactivator recruitment assay in the context of FXR monomer or FXR/RXR heterodimer, with or without RXR agonist 9cRA (Table 2). The results show that heterodimerization with RXR will improve the EC50 of all these ligands. However, when 9cRA is added, differentiated effects of the FXR compounds were observed. Ligands HNC180 and GW4064 have better EC50 in FXR/RXR–LBD dimer; on the other hand, EC50 of HNC143, INT747, and ivermectin decrease to FXR monomer level or even lower. This result suggested that the higher-order nuclear receptor complex structure improves coactivator binding, and the synergistic effect between the two receptors ligands may have ligand selectivity.Table 2EC50 of ligands in the coactivator recruitment assay with monomeric or heterodimeric FXR–LBDReceptorEC50HNC143HNC180GW4064INT747IvermectinμmFXR–LBD0.228 ± 0.0150.043 ± 0.0020.131 ± 0.0330.521 ± 0.0270.468 ± 0.049FXR/RXR-LBD0.086 ± 0.0050.032 ± 0.0010.036 ± 0.0120.359 ± 0.0080.396 ± 0.036FXR/RXR–LBD·9cRA0.211 ± 0.0300.049 ± 0.0210.050 ± 0.0040.843 ± 0.1590.726 ± 0.035 Open table in a new tab Consistent with other RXR heterodimer structures, FXR and RXRα interact via the conserved asymmetric dimer interface, largely comprised of H11 in each monomer, with additional contacts from H7 and H9, loops L8–9 and L9–10. The interactions consist of an intricate network of hydrophobic and polar interactions (Table S2), with H11 of RXRα and FXR forming a coiled coil structure as shown in Fig. 4a. During the protein purification, we found that the isolated FXR–LBD is mostly monomeric, whereas mixing equivalent FXR and RXR LBDs yields essentially heterodimer (data not shown). Comparing the structurally available RXR heterodimers with RXR homodimer, RXR partners differ in the degree of bending and orientation of H11, even though they share similar electrostatic and polar contacts in the heterodimer interface. In the nonpermissive TR/RXR heterodimer, TRβ H11 has a marked shift, resulting in a rotation of H11 and H5 in RXRα and then disruption of the active conformation of RXRα (20Kojetin D.J. Matta-Camacho E. Hughes T.S. Srinivasan S. Nwachukwu J.C. Cavett V. Nowak J. Chalmers M.J. Marciano D.P. Kamenecka T.M. Shulman A.I. Rance M. Griffin P.R. Bruning J.B. Nettles K.W. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers.Nat. Commun. 2015; 6 (26289479)801310.1038/ncomms9013Crossref PubMed Scopus (88) Google Scholar). Although the backbone of the N terminus of H11 in the permissive RXR partners superimpose well with the RXR homodimer, noticeable shifts exist in the C terminus of H11 (Fig. 4b). The differences in the heterodimer interface (especially H11) between the permissive and nonpermissive RXR heterodimer suggest that receptor/ligand-specific bending of H11 also contributes to the cross-dimer signaling. Previous studies have shown that an electrostatic tethering across the dimer interface exists in most RXR heterodimers in the plane of ligand, suggesting a conduit for structural information from the ligand to the partner receptor (22Nettles K.W. Greene G.L. Ligand control of coregulator recruitment to nuclear receptors.Annu. Rev. Physiol. 2005; 67 (15709961): 309-33310.1146/annurev.physiol.66.032802.154710Crossref PubMed Scopus (215) Google Scholar). In our FXR/RXR heterodimer structures, residue Glu434 in RXRα can form a hydrogen bond across the dimer interface with His445 in FXR (Fig. 5d). We then tested several RXR–LBD Glu434 mutations (E434N, E434Q, E434K, and E434A) with WT FXR–LBD in the coactivator binding assay, using RXRα agonist 9cRA and FXR agonist GW4064. The results showed that RXR mutants could affect the signal transduction between the heterodimer (Fig. 5a). When Glu434 in RXR was mutated to Ala, the FXR agonist could hardly increase the ability of RXR to recruit SRC1. Using FXR and RXR mutants, cell-based transfection assay demonstrated that both full-length FXR H445A and RXR E434A mutations decreased the synergistic effect of 9cRA and GW4064 (Fig. 5b). These results indicated that the interaction between the RXR Glu434 and FXR His445 in the heterodimer interface is essential for the synergistic effect. We then looked at whether His445 in FXR is conserved in other RXR partners. Structure-based sequence alignment showed that the presence of basic amino acid (His) at this particular position is unique for FXR. In other nuclear receptors, the residue that forms the electrostatic tethering with Glu434 in RXR corresponds to the position one turn following the His445 in H11 of FXR (Fig. S3). Using SCA, Ranganathan and co-workers (11Shulman A.I. Larson C. Mangelsdorf D.J. Ranganathan R. Structural determinants of allosteric ligand activation in RXR heterodimers.Cell. 2004; 116 (15016376): 417-42910.1016/S0092-8674(04)00119-9Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar) identified a network of 27 coevolved amino acids that are energetically coupled and mediate allosteric signaling in the RXR heterodimers. The mutagenesis study showed that FXR E326A and the corresponding mutations in other permissive partners LXR, PPAR, and Nurr1 exhibit different transcriptional activity in RXR heterodimer. In our GW4064–FXR/9cRA–RXR structure, residue Glu326 in FXR forms salt bridges with and stabilizes Arg441 and Arg436, which participate in the formation of heterodimer interface (Fig. 5d). The E326A mutation would disrupt the interaction network and hinder the allosteric signal transduction from RXR, thereby decreasing the transcriptional activity of FXR. Furthermore, we generated other FXR mutations (R441A and R455S) that belong to the SCA network; each of them decreased the synergistic effect of the two receptor agonists in the cell transactivation assay (Fig. 5c). Taken together, these findings are directly in line with previous findings found in other RXR heterodimers: there are two patches of complementary clustered residues for signal transduction through H11 in FXR–RXR heterodimer. One is through the physically connected AF2 hydrophobic core of the receptor across the dimer interface in the SCA network. The other one connects the dimer interface via an electrostatic tethering across H11 at the plane of the ligands (Fig. S4). We then addressed the question of whether the dimerization interface could be altered depending on the type of bound ligands, thereby providing a structure basis for the allosteric communication between RXR heterodimers and diverse gene activation profiles (23Downes M. Verdecia M.A. Roecker A.J. Hughes R. Hogenesch J.B. Kast-Woelbern H.R. Bowman M.E. Ferrer J.L. Anisfeld A.M. Edwards P.A. Rosenfeld J.M. Alvarez J.G. Noel J.P. Nicolaou K.C. Evans R.M. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR.Mol. Cell. 2003; 11 (12718892): 1079-109210.1016/S1097-2765(03)00104-7Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). In our three FXR–RXR heterodimer structures, all RXRα agonists are 9cRA (Fig. S5). Structure superposition of these complexes showed that the RXR parts superpose well (Fig. 6a), with root-mean-square deviations being 0.460 Å (GW4064–FXR/9cRA–RXR versus HNC143–FXR/9cRA–RXR) and 0.457 Å (HNC180–FXR/9cRA–RXR versus HNC143–FXR/9cRA–RXR) over RXRα Cα atoms. One significant difference is observed in the C terminus of H11 of RXR in HNC143–FXR/9cRA–RXR heterodimer, among which movement of residue" @default.
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• W2894803492 date "2018-11-01" @default.
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• W2894803492 title "Ligand binding and heterodimerization with retinoid X receptor α (RXRα) induce farnesoid X receptor (FXR) conformational changes affecting coactivator binding" @default.
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