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Structural Basis for HNF-4α Activation by Ligand and Coactivator Binding

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m400864200

1083-351X

Karen M. Duda, Young-In Chi, Steven E. Shoelson,

Protein Degradation and Inhibitors

In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4α showed an unusual degree of structural flexibility in the AF-2 domain (helix α12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with α12 fully extended and colinear with α10. By contrast, the second molecule in each homodimer was in a closed conformation with α12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4α as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4α bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4α, coactivator rather than ligand binding locks the active conformation. In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4α showed an unusual degree of structural flexibility in the AF-2 domain (helix α12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with α12 fully extended and colinear with α10. By contrast, the second molecule in each homodimer was in a closed conformation with α12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4α as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4α bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4α, coactivator rather than ligand binding locks the active conformation. As a member of the nuclear receptor family of transcription factors, HNF-4α can be separated into DNA and ligand binding domains (LBD). 1The abbreviations used are: LBD, ligand binding domain; PDB, Protein Data Bank; PEPCK, phosphoenolpyruvate carboxykinase; NR, nuclear receptor; FFA, free fatty acid; RXR, retinoid X receptor. The HNF-4α LBD (residues 140–368) contains distinctive features such as a hydrophobic ligand binding pocket, a dimerization interface, and an AF-2 transactivation domain (residues 360–368) (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). HNF-4α expression is highest in the liver, kidney, and the small and large intestines and lower but detectable in pancreatic β cells and the stomach (2Sladek F.M. Zhong W.M. Lai E. Darnell Jr., J.E. Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (861) Google Scholar). HNF-4α plays critical roles in nutrient transport and metabolism. Its target genes include PEPCK, a major enzyme in the gluconeogenic pathway (3Hall R.K. Scott D.K. Noisin E.L. Lucas P.C. Granner D.K. Mol. Cell. Biol. 1992; 12: 5527-5535Crossref PubMed Scopus (77) Google Scholar), as well as Apo-AI, Apo-II, Apo-IV, Apo-B, Apo-CII, Apo-III, and Apo-E, which are involved in lipid transport and metabolism (4Ladias J.A. Hadzopoulou-Cladaras M. Kardassis D. Cardot P. Cheng J. Zannis V. Cladaras C. J. Biol. Chem. 1992; 267: 15849-15860Abstract Full Text PDF PubMed Google Scholar, 5Kardassis D. Sacharidou E. Zannis V.I. J. Biol. Chem. 1998; 273: 17810-17816Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 6Ktistaki E. Lacorte J.M. Katrakili N. Zannis V.I. Talianidis I. Nucleic Acids Res. 1994; 22: 4689-4696Crossref PubMed Scopus (67) Google Scholar). Targeted deletion studies indicate that HNF-4α is necessary for normal embryonic development (7Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr., J.E. Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (485) Google Scholar, 8Duncan S.A. Nagy A. Chan W. Development. 1997; 124: 279-287Crossref PubMed Google Scholar). Full activity is achieved through the interaction of HNF-4α homodimers with DNA and coactivators, including SRC-1, PGC-1, and GRIP-1. These coactivators contain nuclear receptor (NR) boxes comprised of LXXLL motifs. The sequence on either side of the NR boxes imparts specificity to binding that allows the same coactivator to have different affinities for different nuclear receptors. The ability of NRs to form various coactivator complexes provides a mechanism for activating discrete transcriptional processes (9Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1108) Google Scholar, 10Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Crossref PubMed Google Scholar, 11Voegel J.J. Heine M.J. Tini M. Vivat V. Chambon P. Gronemeyer H. EMBO J. 1998; 17: 507-519Crossref PubMed Scopus (432) Google Scholar). HNF-4α has also been linked to nutrient metabolism through interactions with the coactivator PGC-1, potentially providing a driving force for gluconeogenesis (12Rhee J. Inoue Y. Yoon J.C. Puigserver P. Fan M. Gonzalez F.J. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4012-4017Crossref PubMed Scopus (476) Google Scholar). Mutations in hnf-4α are associated with a rare monogenic form of diabetes referred to as MODY1 (maturity-onset diabetes of the young). MODY1 patients are characterized by an insulin secretion defect in the face of normal insulin sensitivity (13Malecki M.T. Yang Y. Antonellis A. Curtis S. Warram J.H. Krolewski A.S. Diabetes Med. 1999; 16: 193-200Crossref PubMed Scopus (30) Google Scholar). Recent genetic studies have linked polymorphisms in or near the islet-specific P2 promoter of hnf-4α to type 2 diabetes (14Love-Gregory L.D. Wasson J. Ma J. Jin C.H. Glaser B. Suarez B.K. Permutt M.A. Diabetes. 2004; 53: 1134-1140Crossref PubMed Scopus (202) Google Scholar, 15Silander K. Mohlke K.L. Scott L.J. Peck E.C. Hollstein P. Skol A.D. Jackson A.U. Deloukas P. Hunt S. Stavrides G. Chines P.S. Erdos M.R. Narisu N. Conneely K.N. Li C. Fingerlin T.E. Dhanjal S.K. Valle T.T. Bergman R.N. Tuomilehto J. Watanabe R.M. Boehnke M. Collins F.S. Diabetes. 2004; 53: 1141-1149Crossref PubMed Scopus (240) Google Scholar), suggesting that reduced HNF-4α activity in pancreatic β cells might also be associated with and possibly predispose to the development of this prevalent disorder. Nuclear receptor LBDs are found in two distinct conformations. The difference between forms is in the position of helix α12, often referred to as the AF-2 domain. In the first conformation, α12 is extended colinearly with helices α10 and α11, leaving the ligand binding pocket accessible and the LBD in what is referred to as the "open" conformation. Because this structure does not allow the binding of coactivator molecules, it is also considered to be the inactive form. In the second conformation, helix α12 folds back onto the LBD to seal off the ligand binding pocket. Because this "closed" form interacts with coactivators, it is considered to be the active state of the NR. Structural comparisons between unliganded (apodomain) and ligand-bound and coactivator-bound forms of various NRs have led to the supposition that it is ligand binding that activates NRs by converting molecules in the open inactive conformation to an all closed active state (16Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar, 17Renaud J.P. Moras D. Cell Mol. Life Sci. 2000; 57: 1748-1769Crossref PubMed Scopus (210) Google Scholar). Our previously reported crystal structure of HNF-4α challenges this hypothesis (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). One major finding from both the HNF-4α and HNF-4γ structures was the serendipitous presence of fatty acids in their LBDs, suggesting that these might be endogenous ligands (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Wisely G. Miller A. Davis R. Thornquest A. Johnson R. Spitzer T. Sefler A. Shearer B. Moore J. Miller A. Willson T. Williams S. Structure. 2002; 10: 1225-1234Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). However, only half of the fatty acid-bound molecules in the HNF-4α structure adopted the closed conformation expected for the activated state. The second molecule in each homodimer was in a conformation expected for the inactive state with helix α12 fully extended and colinear with helix α10. This finding suggested that fatty acid binding might be necessary but not sufficient for activating HNF-4α. To better understand the structural mechanism for ligand- and coactivator-mediated activation of HNF-4α, we have crystallized its LBD bound to both fatty acid ligand and a coactivator peptide sequence derived from SRC-1. Protein Production and Crystallization—The protein boundaries for expression of the human HNF-4α LBD were defined by our previous structure of the rat apodomain (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) (the LBDs of rat and human HNF-4α are 97% identical). DNA encoding the LBD of human HNF-4α (residues 140–382) was subcloned into the pET 28a vector (Novagen) by PCR. Protein was expressed in Escherichia coli BL21(DE3) (Invitrogen) cells and isolated from lysates using Talon cobalt affinity resin (Clontech). The His6 affinity tag was cleaved using bovine thrombin (10 units/ml), and the protein was further purified by ion-exchange chromatography (Mono Q fast protein liquid chromatography). A 3-fold molar excess of SRC-1 peptide (SSLTERHKILHRLLQEGSPS, residues 681–700) was incubated with the protein for 1 h at 4 °C prior to concentration (15 mg/ml). Crystals were obtained at room temperature by the vapor diffusion method in 20-μl drops containing equal volumes of protein/peptide complex and crystallization buffer (0.1 m Hepes, pH 7.0, 0.7 m sodium/potassium tartrate and 0.01 m dithiothreitol). Crystals belonging to the space group P41212 reached maximum dimensions of 0.3 × 0.2 × 0.2 mm within 5 days. Data Collection and Structure Determination—Diffraction data were collected at beamline X12C of the National Synchrotron Light Source (Brookhaven, NY). Oscillation images (every 1°) were collected at 100 K, and the data were processed using the DENZO HKL software package (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The structure was determined by molecular replacement using the program MOLREP (20Hu J. Liu J. Ghirlando R. Saltiel A.R. Hubbard S.R. Mol. Cell. 2003; 12: 1379-1389Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and our previous structure of HNF-4α in complex with fatty acid as a search model (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Each asymmetric unit contains one ternary complex comprising the LBD of human HNF-4α, one fatty acid, and one SRC-1 peptide. The initial R value was 0.48 with a correlation coefficient of 0.57. The subsequent σ -weighted 2Fo-Fc map after rigid body refinement clearly revealed density corresponding to the bound SRC-1 peptide that was not present in the search model. The model was refined using an amplitude-based maximum-likelihood target in the program CNS (21Brunger 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. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), applying bulk solvent corrections and individual B-factor corrections at the final stage. The data and refinement statistics are summarized in Table I. Graphics were generated using the program O (22Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar).Table IData and refinement statisticsNative Data StatisticsUnit cella = b = 81.49 Å, c = 104.71 ÅWavelength (Å)0.9790Resolution (Å)29.9–2.1Unique reflections20,187Redundancy12.10Completeness (%)aData in parentheses refer to the highest resolution shell95.1 (76.7)I/σ(I)aData in parentheses refer to the highest resolution shell32.68 (3.59)Rsym (%)aData in parentheses refer to the highest resolution shell,bRsym = Σ|Iobs — 〈I〉|/Σ〈I〉, calculated for all data4.7 (22.5)Refinement StatisticsResolution (Å)20–2.1Reflections19,974R-factor (%)22.6Rfree, 5% of data (%)26.8Non-H atoms per asymmetric unitProtein/peptide1936Fatty acid ligand16Water137〈B〉cAverage temperature factor42.82 Å2R.m.s. deviationdR.m.s., root mean squareBond length0.006 ÅBond angles1.08°Dihedral angles18.92°Improper angles0.74°a Data in parentheses refer to the highest resolution shellb Rsym = Σ|Iobs — 〈I〉|/Σ〈I〉, calculated for all datac Average temperature factord R.m.s., root mean square Open table in a new tab Domain Architecture—The LBD of human HNF-4α is dimeric in the crystals with one protein molecule in each asymmetric unit. It adopts the canonical nuclear receptor LBD fold containing nine α helices and two β strands (Fig. 1A). The helices were numbered according to conventional NR nomenclature as we had previously numbered the helices in the rodent protein (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Helix α2, which is variably present in NR LBDs, is not present in HNF-4α. α6 consists of only one helical turn, and because α10 and α11 are contiguous, this helix is referred to simply as α10. The nine α helices are organized into three layers within the helical sandwich. Helices α4, α5, α8, and α9 form the central layer. Helices α1 and α3 comprise the outer layer at one side of the central layer, and helix α10 forms the opposite outer layer. Helix α12 is separate from α10, consistent with the HNF-4α LBD being in the active state and bound to coactivator. The dimerization interface, comprising residues from α9 and α10, lies along a plane of 2-fold crystallographic symmetry. At 2.1-Å resolution, the amino acid side chains are well defined in the electron density map with the exception of side chains from residues of the α1-α3 loop. There are two obvious differences between the structures of the binary complex of the FFA-bound rat domain (Fig. 1B) reported earlier (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar) and the ternary complex of the human domain reported here (Fig. 1A). The first is the presence of the SRC-1 peptide in one structure and not the other, which provides the opportunity to determine the effects of coactivator binding both on global structure and more specifically on the orientation of α12 (the AF-2 domain). The second difference, linked to the first, addresses the issue of open and closed configurations and what they mean. The LBD adopts two distinct conformations in the binary complex, despite both molecules being bound to fatty acid ligand (Fig. 1, B and C) (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The first molecule is in an open conformation in which α10 and α12 are contiguous and colinear. This long helix is fully extended. The second molecule of each homodimer adopts a closed conformation with α12 situated against the body of the LBD. In the closed state, a hydrophobic patch on α12 (Leu-360, Leu-361, Met-364, and Leu-365) affixes it via hydrophobic interactions to the body of the domain. Residues from α3 (Met-182, Ala-183, Leu-186, Leu-187, Leu-189, and Val-190) and α4 (Leu-211, Ala-215, Gly-216, and Leu-219) form a complementary patch on the body of the domain. Both molecules in the ternary LBD complex (HNF-4α·FFA·SRC-1) adopt identical closed conformations (Fig. 1A). This structure is indistinguishable from that of the molecule in the closed conformation of the binary complex (Fig. 1C), yielding an root mean square deviation value of 0.57 Å when all of the Cα atoms of the two structures are superimposed. We have concluded from these comparisons that ligand (FFA) binding is conformationally permissive, because it facilitates the formation of the activated closed state but does not lock it. Coactivator binding, on the other hand, is restrictive because it apparently locks FFA-bound HNF-4α into the activated closed state. HNF-4α/Fatty Acid Interactions—X-ray crystal structures of rat HNF-4α and human HNF-4γ indicated that both of these proteins bind fatty acids as natural ligands (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Wisely G. Miller A. Davis R. Thornquest A. Johnson R. Spitzer T. Sefler A. Shearer B. Moore J. Miller A. Willson T. Williams S. Structure. 2002; 10: 1225-1234Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Because fatty acids incorporated spontaneously into the ligand binding pockets of the proteins during bacterial expression and because it was not possible to remove the fatty acids without at least partially denaturing the proteins, we had hypothesized that ligands, presumably fatty acids, might be critical to the stably folded domains (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 18Wisely G. Miller A. Davis R. Thornquest A. Johnson R. Spitzer T. Sefler A. Shearer B. Moore J. Miller A. Willson T. Williams S. Structure. 2002; 10: 1225-1234Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Our new structure shows that human HNF-4α similarly sequesters bacterial fatty acids during protein production. Fatty acids are once again present in all of the molecules of the structure, despite not having been added intentionally. The ligand binding pocket forms a narrow channel that is lined almost exclusively with side chains of hydrophobic residues (Fig. 2). The side chain of Arg-226 at the base of the binding pocket is the exception. Considerably smaller than the ligand binding pockets of other reported NR LBDs, HNF-4α has a cavity area of 370 Å3 as calculated by the program Voidoo (23Kleywegt G.J. Jones T.A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 178-185Crossref PubMed Scopus (988) Google Scholar). This is the approximate molecular volume of a long chain fatty acid. The fatty acids bound to our structure are anchored in the ligand binding pocket via an interaction between the fatty acid headgroup and the side chain of Arg-226. Both of the oxygen molecules in the fatty acid headgroup interact with the arginine guanidinium group, and one oxygen interacts as well with the backbone NH group of Gly-237. The ligand interaction is further stabilized by hydrophobic and Van der Waals interactions between the fatty acid carbon chain and residues lining the pocket. Residues that participate in these interactions include Ile-175, Val-178, Cys-179, Met-182, Leu-219, Leu-220, Arg-223, Leu-236, Met-252, Val-255, Ile-259, Met-342, Ile-346, Ile-349, and Ile-357 (Fig. 2). Because our previous structure was of the binary FFA-bound LBD complex and our new structure is of the ternary HNF-4α·FFA·SRC-1 complex, we are in a position to compare ligand binding pockets in the absence and presence of bound coactivator. Improved electron density for the ternary complex allowed visualization of 14 carbons of the fatty acid, as opposed to the 12 carbons seen in the binary complex (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Improved electron density similarly facilitated the identification of additional water molecules in the ligand binding pocket of the ternary complex. Otherwise, there appear to be no significant differences (Fig. 2). As noted previously, backbone Cα atoms are superimposable. Amino acid side chains that line the pockets are in similar positions in the two structures and maintain the same interactions, including the salt bridge between the side chain of Arg-226 and the fatty acid head. The trivial differences in fatty acid binding between the two structures can thus be accounted for by the enhanced resolution of the ternary structure, solved at a 2.1-Å resolution as opposed to a 2.8-Å resolution. HNF-4α/SRC-1 Interactions—As a recurring structural theme for nuclear receptors, bound coactivators form α-helices with hydrophobic side chains of the LXXLL motif directed inward to interact with the LBDs (24Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1778) Google Scholar). The HNF-4α·FFA·SRC-1 complex recapitulates this mode of interaction (Fig. 3). Of 20 residues in the crystallized SRC-1 peptide, 14 at the carboxyl terminus are well defined. The amino-terminal six residues are not visualized in the electron density map. Eight residues (ILHRLLQEG) encompassing the LXXLL motif form >2 full turns of an α-helix. The HNF-4α/SRC-1 interaction is dominated by hydrophobic interactions involving the leucine residues of the LXXLL motif and the canonical "charge clamp" created by hydrogen bonds between backbone atoms and the side chains of invariant residues Glu-363 of α12 and Lys-194 of α3. The interaction with Gly-363 requires that α12 be folded into place in the ligand binding pocket and thus mandates that coactivator binding occur only with the closed LBD conformation. The hydrophobic face of the LXXLL helix is packed into a hydrophobic pocket created by residues in α3 (Leu-187, Val-190, and Lys-194), α3-α4 loop (Phe-199), α4 (Leu-211), and α12 (Glu-363). The charge clamp orients the LXXLL motif into place, which explains why modes of interactions are well conserved between NRs and coactivators. Subtler interactions are thought to govern specificity and relative affinity of a given NR for different coactivator LXXLL motifs. These may be mediated by flanking residues at the carboxyl-terminal end of the LXXLL helix (25McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Crossref PubMed Scopus (529) Google Scholar, 26Darimont 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 (832) Google Scholar). In our structure, the amino-terminal flanking region of the SRC-1 peptide is disordered, whereas the carboxyl-terminal flanking region is well defined but lacks specific interactions with the LBD. Helix α12 Structural Changes—Because ligand-bound HNF-4α adopts both closed (active) and open (inactive) conformations (1Dhe-Paganon S. Duda K. Iwamoto M. Chi Y.I. Shoelson S.E. J. Biol. Chem. 2002; 277: 37973-37976Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), whereas the SRC-1-bound LBD adopts only the closed conformation, α12 does not appear to be a ligand-dependent switch that upon ligand binding triggers a structural rearrangement, providing a surface for coactivator binding (27Renaud J.P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1034) Google Scholar, 28Nolte 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 (1700) Google Scholar). Moreover, α12 does not participate in direct interactions with the ligand as is seen in the interactions of other NRs with many synthetic ligands. Therefore, it appears that both ligand and co-activator binding are required to lock HNF-4α in the closed and active state. Comparisons between Unliganded NRs and Binary and Ternary Complexes—The first apodomain structure to be determined was that of RXR (29Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1067) Google Scholar). Comparisons between the apodomain and structures of the binary ligand/RXR and ternary ligand/coactivator/RXR complexes formed the basis for developing a global theory regarding structural determinants in NR activation. The theory predicted that apodomains are open and that ligand binding induces a conformational transition that stabilizes a closed active state with α12 (AF-2) folded against the body of the domain to form a coactivator binding surface (16Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar, 17Renaud J.P. Moras D. Cell Mol. Life Sci. 2000; 57: 1748-1769Crossref PubMed Scopus (210) Google Scholar). The theory further predicted that although coactivator binding might further stabilize the closed conformation, it would not produce additional conformational changes. Because HNF-4α does not fit this scheme, we have re-examined the many structures that have now been solved. Of the four published apodomain structures (29Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1067) Google Scholar, 30Watkins R.E. Wisely G.B. Moore L.B. Collins J.L. Lambert M.H. Williams S.P. Willson T.M. Kliewer S.A. Redinbo M.R. Science. 2001; 292: 2329-2333Crossref PubMed Scopus (719) Google Scholar, 31Uppenberg J. Svensson C. Jaki M. Bertilsson G. Jendeberg L. Berkenstam A. J. Biol. Chem. 1998; 273: 31108-31112Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 32Gampe Jr., R.T. Montana V.G. Lambert M.H. Wisely G.B. Milburn M.V. Xu H.E. Genes Dev. 2000; 14: 2229-2241Crossref PubMed Scopus (127) Google Scholar), RXR is the only one with α12 in the open conformation (Fig 4A). This indicates that, in the absence of ligand, α12 is conformationally flexible and can adopt both open and closed configurations. The predominance of the closed form seen in structural studies, in addition to data from thermal melt studies, suggests that the closed form of the apodomain may be energetically favored (33Watkins R.E. Davis-Searles P.R. Lambert M.H. Redinbo M.R. J. Mol. Biol. 2003; 331: 815-828Crossref PubMed Scopus (194) Google Scholar). Three structures of binary complexes of LBDs bound to naturally occurring ligands are shown in Fig. 4B. RXRα is bound to 9-cis-retinoic acid (PDB 1FBY) (16Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar), retinoid acid-related orphan receptor α is bound to cholesterol (PDB 1N83) (34Kallen J.A. Schlaeppi J.M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), and the androgen receptor is bound to dihydrotestosterone (PDB 1I37) (35Sack J.S. Kish K.F. Wang C. Attar R.M. Kiefer S.E. An Y. Wu G.Y. Scheffler J.E. Salvati M.E. Krystek Jr., S.R. Weinmann R. Einspahr H.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4904-4909Crossref PubMed Scopus (391) Google Scholar). LBDs in previously reported structures of binary ligand-bound complexes adopt the active closed conformation, supporting the theory that ligand binding serves as the activating switch. However, the finding that binary HNF-4α·FFA complexes adopt both open and closed conformations in our recently solved structure runs counter to the theory. In reexamining the structural basis for activation, found that many endogenous ligands in published structures do not interact with α12 directly (16Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. 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The coactivator charge clamp located on either side of the LXXLL motif interacts directly with α12 in each of these structures. Thus, coactivator binding requires that the LBD be closed and, because the LBD must remain closed as long as the coactivator is bound, this essentially "locks" the LBD in an active conformation. We conclude for HNF-4α that although ligand binding and coactivator binding both stabilize the LBD, it is coactivator binding that locks it in the active state. As far as we can tell from available structures of other LBDs bound to endogenous ligands and coactivators, the same holds, i.e. the binding of natural ligands and coactivators stabilize the LBD but coactivator binding locks the active state. Synthetic ligands with bulky appendages that interact with α12 may have the added ability to lock LBDs in the active conformation. We thank the staff at the National Synchrotron Light Source beamline X12C, Brookhaven National Laboratory for valuable assistance.