Crystal Structure of the Ligand-binding Domain of the Ultraspiracle Protein USP, the Ortholog of Retinoid X Receptors in Insects

Artigo Acesso aberto Revisado por pares

Crystal Structure of the Ligand-binding Domain of the Ultraspiracle Protein USP, the Ortholog of Retinoid X Receptors in Insects

2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês

10.1074/jbc.m008926200

ISSN

1083-351X

Autores

Isabelle M. L. Billas, Luc Moulinier, Natacha Rochel, Dino Moras,

Tópico(s)

Antioxidant Activity and Oxidative Stress

Resumo

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides1G2N. The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides1G2N. ultraspiracle protein ecdysone receptor retinoid X receptor nuclear receptor activation function 2 estrogen receptor ligand-binding domain juvenile hormone 9-cis-retinoic acid retinoic acid receptor root mean square deviation The Ultraspiracle protein (USP),1 an orphan nuclear receptor, is the insect ortholog of the vertebrate retinoid X receptor (RXR). Like RXR, it belongs to the superfamily of nuclear receptors (NR) (1Laudet V. Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar), which are intracellular receptors regulating target gene expression upon binding of small, hydrophobic molecules like steroids, retinoids, thyroid hormones, and vitamin D3. In insects, steroid hormones, the ecdysteroids, control insect development, molting, metamorphosis, and reproduction (2Lezzi M. Bergman T. Mouillet J.-F. Henrich V.C. Arch. Insect Biochem. Physiol. 1999; 41: 99-106Crossref Scopus (34) Google Scholar, 3Nijhout H.F. Insect Hormones. Princeton University Press, Princeton, NJ1994Google Scholar, 4Thummel C.S. Trends Genet. 1996; 12: 306-310Abstract Full Text PDF PubMed Scopus (411) Google Scholar). The weak binding of 20-hydroxyecdysone, the biologically active ecdysteroid for most insects, to the ecdysone nuclear receptor (EcR) is dramatically stimulated by addition of USP, resulting in an affinity in the nanomolar range (5Yao T.-P. Forman B.M. Jiang Z. Cherbas L. Chen J.-D. McKeown M. Cherbas P. Evans R.M. Nature. 1993; 366: 476-479Crossref PubMed Scopus (776) Google Scholar). In fact, the functional ecdysteroid receptor is formed by the heterodimer EcR/USP, as demonstrated in vitroby DNA binding, ligand binding, and transactivation assays (6Wang S.-F. Miura K. Misiceks R.J. Segraves W.A. Raikhel A.S. J. Biol. Chem. 1998; 273: 27531-27540Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) andin vivo on fly usp mutants (7Hall B.L. Thummel C.S. Development ( Camb. ). 1998; 125: 4709-4717PubMed Google Scholar). Like its vertebrate homolog, USP can heterodimerize with several vertebrate NRs (5Yao T.-P. Forman B.M. Jiang Z. Cherbas L. Chen J.-D. McKeown M. Cherbas P. Evans R.M. Nature. 1993; 366: 476-479Crossref PubMed Scopus (776) Google Scholar), and in insects, it forms heterodimeric complexes with EcR and also with at least another NR, DHR38 (8Sutherland J.D. Kozlova T. Tzertzinis G. Kafatos F.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7966-7970Crossref PubMed Scopus (132) Google Scholar). For vertebrates, the heterodimerization of RXR with several NRs allows the modulation of their affinity for genomic response elements (9Chen J.Y. Clifford J. Zusi C. Starrett J. Tortolani D. Ostrowski J. Reczek P.R. Chambon P. Gronemeyer H. Nature. 1996; 382: 819-822Crossref PubMed Scopus (182) Google Scholar, 10Vivat V. Zechel C. Wurtz J.-M. Bourguet W. Kagechika H. Umemiya H. Shudo K. Moras D. Gronemeyer H. Chambon P. EMBO J. 1997; 16: 5697-5709Crossref PubMed Scopus (114) Google Scholar, 11Minucci S. Leid M. Toyama R. Saint-Jeannet J.P. Peterson V.J. Horn V. Ishmael J.E. Bhattacharyya N. Dey A. Dawid I.B. Ozato K. Mol. Cell. Biol. 1997; 17: 644-655Crossref PubMed Scopus (148) Google Scholar). Unlike its vertebrate homolog RXR for which a ligand is known, the 9-cis-retinoic acid (9-cis-RA), no hormone ligand has been identified for USP up to now. Juvenile hormone (JH), an esterified sesquiterpene, has been put forward as the candidate hormone ligand of USP. In fact, it has been known for a long time that JH prevents metamorphosis by modulating the ecdysteroid action at the outset of the ecdysteroid rise for the molt (3Nijhout H.F. Insect Hormones. Princeton University Press, Princeton, NJ1994Google Scholar, 12Hall B.L. Am. Zool. 1999; 39: 714-721Crossref Scopus (9) Google Scholar, 13Riddiford L.M. Hiruma K. Lan Q. Zhou B. Am. Zool. 1999; 39: 736-746Crossref Scopus (58) Google Scholar, 14Riddiford L.M. Receptor. 2000; 3: 203-209Google Scholar). The hypothesis that JH might act through a NR relies on its chemical analogy with the vertebrate terpenes, represented by the retinoic acid. The idea that USP could be the receptor of JH or any of its derivatives raises the attractive possibility that JH might directly modulate the activity of the EcR/USP complex (15Buszczak M. Segraves W.A. Curr. Biol. 1998; 8: R879-R882Abstract Full Text Full Text PDF PubMed Google Scholar). In addition, some evidence was given that JH can bind to USP and stimulate oligomerization of USPin vitro and in yeast cells (16Jones G. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13499-13503Crossref PubMed Scopus (239) Google Scholar). However, this is largely debated and still awaits further substantiation (2Lezzi M. Bergman T. Mouillet J.-F. Henrich V.C. Arch. Insect Biochem. Physiol. 1999; 41: 99-106Crossref Scopus (34) Google Scholar). In particular, the dissociation constant for binding of JH to USP was measured to be about 0.5 μm (16Jones G. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13499-13503Crossref PubMed Scopus (239) Google Scholar). Compared with the typical affinity of hormones for their nuclear receptor (in the nanomolar range), this low affinity questions whether such concentrations might be meaningful at a physiological level, where other cellular molecules might compete with JH for USP binding. NRs are modular proteins possessing a highly conserved DNA-binding domain and a moderately conserved ligand-binding domain (LBD) (17Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (682) Google Scholar). The LBD confers specificity to ligand binding and is responsible for ligand-dependent transactivation. Crystallographic investigations of the LBD structures of several NRs indicate a conserved fold described as an antiparallel α-helical sandwich (18Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar) composed of 11 α-helices (H1, H3–H12) and two short β-strands (s1–s2). Most importantly, the crystal structures of the LBDs of unliganded (apo) (18Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar) and liganded RXR (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar) and of other liganded NRs (for a review, see Refs. 20Egea P.F. Klaholz B.P. Moras D. FEBS Lett. 2000; 476: 62-67Crossref PubMed Scopus (86) Google Scholar and 21Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (707) Google Scholar) have allowed us to gain insight into the molecular mechanisms that underlie the dramatic structural reorganization that accompanies the ligand binding to the LBD. This conformational rearrangement mostly affects the N-terminal part of H3, H11, and H12, which carries the autonomous activation function (AF-2) (18Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar, 19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar, 22Renaud J.-P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar). Upon binding of an agonist ligand, the corepressors are released, and the LBD adopts a unique conformation that generates an interaction surface for the coactivators, which then recruit multiprotein complexes and lead to the activation of responsive genes (23Torchia J. Glass C. Rosenfeld M.G. Curr. Opin. Cell Biol. 1998; 10: 373-383Crossref PubMed Scopus (512) Google Scholar). In contrast, antagonist ligands induce a transconformation of the LBD that does not allow binding of coactivators. Several antagonist conformations have been observed that can be considered variations around a common theme (24Brzozowski A.M. Pike A.C.W. Dauter Z. Hubbard R.E. Bonn T. Engström O. Öhman L. Greene G.L. Gustafsson J.-A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2945) Google Scholar, 25Shiau 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 (2252) Google Scholar, 26Bourguet 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 (353) Google Scholar, 27Pike A.C.W. Brzozowski A.M. Hubbard R.E. Bonn T. Thorsell A.-G. Engström O. Ljunggren J. Gustafsson J.-A. Carlquist M. EMBO J. 1999; 18: 4608-4618Crossref PubMed Scopus (912) Google Scholar). In these cases, the activation helix H12 lies in a groove similar to the binding site of the helical NR-box module of nuclear coactivators (the so called antagonist groove) (25Shiau 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 (2252) 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 (1689) Google Scholar, 29Darimont 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 (831) Google Scholar). In the case of partial agonist/antagonist ligands, helix H12 also lies in this groove, even though the ligands do not sterically preclude the agonist position of H12. However, in contrast to the full antagonist ligands, a weak but clear transcriptional AF-2 activity is reported in the presence of the corresponding ligand as would be in the presence of an AF-2 full agonist ligand. Partial agonist/antagonist AF-2 conformations were reported for ERβ/genistein (27Pike A.C.W. Brzozowski A.M. Hubbard R.E. Bonn T. Thorsell A.-G. Engström O. Ljunggren J. Gustafsson J.-A. Carlquist M. EMBO J. 1999; 18: 4608-4618Crossref PubMed Scopus (912) Google Scholar) and RXRα/oleic acid (26Bourguet 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 (353) Google Scholar) LBD structures. In this paper, we report the crystal structure of the LBD of the orphan nuclear receptor USP of the lepidopteran H. virescens(hvUSP) at 1.65-Å resolution. The ligand-binding domain of hvUSP adopts an antagonist AF-2 conformation that could be driven by the presence of an unexpected ligand in its ligand-binding cavity. Important structural discrepancies with its vertebrate homolog RXR are observed. In particular, the connecting loop between helices H1 and H3 (L1–3) turns out to be a key element of this receptor in its actual conformation. This structural element sterically precludes the agonist AF-2 conformation and stabilizes an unusual antagonist conformation. The nature of the ligand observed in the crystal structure of USP LBD raises fundamental questions about the biological significance of similar molecules for insect endocrinology. The H. virescens USP (hvUSP) LBD (residues Val-205 to Met-466) was cloned as a N-terminal His6-tagged fusion protein in a pET-15b expression vector and overproduced in the Escherichia coliBL21(DE3) strain. Cells were grown in 2× LB medium at 37 °C and subsequently induced for 2 h with 0.8 mmisopropyl-β-d-thiogalactopyranoside at 24 °C. Purification procedures include an affinity chromatography step on a cobalt chelating column followed by a gel filtration on a Superdex 200 16/60 column. After tag removal by thrombin digestion, protein was further purified by gel filtration. A homogeneous monomeric species was observed in solution. Purity and homogeneity of the protein were assessed by SDS and native polyacrylamide gel electrophoresis, denaturant, and native electrospray ionization mass spectrometry and dynamic light scattering. Crystallization experiments were carried out at 4 °C with the hanging-drop vapor diffusion method. The protein concentration was 3–9 mg/ml. Crystals of 200 × 200 × 400 μm3 were grown in about 10 days from a solution containing 10% polyethylene glycol 4000, 50 mm Tris (pH = 7.5), 100 mm NaCl, 5 mmdithiothreitol equilibrated against a reservoir containing 20% polyethylene glycol 4000 and 100 mm Tris (pH = 7.5). Crystals belong to the tetragonal P4322 space group, with one monomer per asymmetric unit. The unit cell parameters area = 58.21 Å, b = 58.21 Å,c = 144.69 Å, and α = β = γ = 90°. The solvent content amounts to 32%, and the estimated B factor by Wilson plot is 27 Å2. Crystals were flash-frozen in liquid nitrogen after a short dip in a solution containing 10% glycerol as cryoprotectant. The native data set was collected on a single crystal on beamline ID14-EH2 at European Synchrotron Radiation Facility (Grenoble, France). Data were processed (Table I) using HKL programs (30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38556) Google Scholar). The crystal structure was solved by molecular replacement (31Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5028) Google Scholar) using a partial structure of hsRXRα (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar) as a search model. The weak solution was obtained with a correlation of 24.8% andR free = 54.5% after rigid-body refinement. The phasing power of the model was low and required numerous manual building cycles using O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar). The wARP method (33Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (480) Google Scholar) was used as a tool for checking the correctness of the partially built structures. Refinement was performed with CNS (34Brünger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grosse-Kuntstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice M.L. Simonson T. Warre G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16963) Google Scholar) using a maximum likelihood target and bulk solvent correction. Cycles of manual model building and least square minimization followed by simulated annealing and individual isotropic B factor refinement led to the final model. Solvent molecules were located in a F o− F c map contoured at 3ς. The final model, refined to 1.65 Å resolution, comprises 246 residues, 259 water molecules, and one ligand molecule. A large part of the connection loop between helix H5 and the entry of the β-sheet (residues 306–315) as well as the C-terminal extension prolonging H12 (residues 459–466) could not be modeled due to poor electron density in these regions. The quality of the final model (Table I) was monitored with Procheck (35Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar).Table ICrystallographic analysisData collection and phasingX-ray sourceID14-EH2 at ESRFWavelength (Å)0.966Resolution (Å)20.0–1.65Unique reflections30676Completeness (%)99.2Multiplicity7.6R sym(I) (last shell) (%) 1-aR sym(I) = Σhkl Σi ‖I hkl,i − < I hkl> ‖/Σhkl Σi ‖ I hkl,i‖ with the mean intensity of the multipleI hkl,i observations for symmetry-related reflections.4.0 (22.4)I/ς38.2 (4.5)RefinementR free(%) 1-bR free = Σhkl ε T‖F obs −F calc‖/Σhkl ε T‖F obs‖ the test set T includes 10% of the data.24.4Rcryst (%) 1-cR cryst = Σhkl‖F obs −F calc‖/Σhkl‖F obs.21.0Non-hydrogen atomsProtein1998Ligand49Water molecules259R.m.s.d. on bond length (Å)0.01R.m.s.d. on bond angles (°)1.4AverageB-factor for non-hydrogen atoms (Å2)Protein29.7Ligand47.6Water43.8Ramachandran plotMost favored regions91.9Additional allowed regions7.7Generously allowed regions0.51-a R sym(I) = Σhkl Σi ‖I hkl,i − < I hkl> ‖/Σhkl Σi ‖ I hkl,i‖ with the mean intensity of the multipleI hkl,i observations for symmetry-related reflections.1-b R free = Σhkl ε T‖F obs −F calc‖/Σhkl ε T‖F obs‖ the test set T includes 10% of the data.1-c R cryst = Σhkl‖F obs −F calc‖/Σhkl‖F obs. Open table in a new tab Time-of-flight mass spectrometry was utilized either using the electrospray technique (electrospray ionization) in native and denaturating conditions or the fast atom bombardment technique. Results showed that a heterogeneous mass distribution around 740 ± 50 Da was added to the peak corresponding to the pure protein (30.2 kDa), and the major peak corresponds to a mass of 745 ± 3 Da. The fatty acid content of the phospholipid molecules was obtained by organic solvent extraction. The analysis shows that more than 95% of the fatty acids are composed of C18:1 (40%), C16:0 (34%), C16:1 (22%); and trace amounts of C17:0, C18:0, C18:1, and C18:2 are detected as well. In the case of phosphatidylglycerol molecules, the major species detected by Time-of-flight mass spectrometry (745 ± 3 Da) correspond to a molecule with a tail made of C16:0 and C18:1 esterified fatty acids, fully consistent with the results of the organic solvent extraction and with the electron density maps. The present crystallographic investigations of hvUSP were restricted to the LBD, which comprises 264 residues starting at Val-205. The sequence of the hvUSP LBD is shown in Fig. 1 and is aligned to the sequences of other USP LDBs of the insect orders Lepidoptera and Diptera and to sequences of RXR of isotypes α, β, and γ. The sequences of USP LBDs altogether are rather well conserved with respect to those of RXR LBDs (between 34 and 42% sequence identity). However, while the conservation is highly pronounced within the whole RXR family, the USP sequences are highly conserved only inside the lepidopteran family (83–87% sequence identity), those of the dipteran family being much less conserved when compared with each other (between 46 and 54% sequence identity) and to the sequences of the lepidopteran USP LBDs (between 40 and 49% sequence identity). On the basis of this sequence alignment, the secondary structure elements, 11 helices and a β-sheet can be predicted for USP LBDs using the canonical structure of NR LBDs (17Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (682) Google Scholar). The crystal structure of hvUSP LBD supports these predictions, and the secondary structure elements are represented schematically in Fig. 1, together with those of hsRXRα LBD. As readily shown in this figure, the helix H3 of hvUSP LBD is one turn longer compared with its counterpart in the RXRα crystal structure. This figure also indicates that most of the conserved residues between lepidopteran USPs and RXRs are located in the helices, in particular in those forming the core of the LBD as well as within the signature region (17Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (682) Google Scholar). Divergence between USPs and RXRs is observed mainly for two loops that connect helix H5 to the β-turn (s1) (H5-s1) and helix H1 to helix H3 (L1–3). The loop H5-s1 is longer for USPs than for RXRs. Its length also varies inside the USP family, and it shows rather poor sequence conservation. On the other hand, the length of L1–3 is rather similar for USPs and RXRs. Its sequence is poorly conserved between the two families. However, it is highly conserved inside the family of lepidopteran USPs to which hvUSP belongs. This is remarkable, because within the whole NR superfamily L1–3 is usually found to be extremely variable in length and in sequence, consistent with its nature of a rather flexible and loosely structured region. In contrast, the crystal structure of hvUSP LBD presented here shows that L1–3 behaves as a rather stiff region due to strong interactions with several key secondary structure elements of the LBD. The overall architecture of USP LBD exhibits the canonical NR fold (18Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar) with 11 α-helices (H1, H3–H12) and two short β-strands (s1–s2). In the following, the structure of USP LBD will be compared with two other crystal structures that bear the major features of NRs and are closely related to it: the agonist-bound (holo) RXRα (hsRXRα/9-cis-RA) (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar) (Fig.2 A) and the RXRα in an antagonist conformation (msRXRα/oleic acid) (26Bourguet 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 (353) Google Scholar) (Fig. 2 B) LBDs. The superimposition of the USP LBD to the structure of holo-RXRα LBD was done by a least square fit (using the LSQ options of O (32Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar)). Overall, the secondary structure elements of the USP LBD superimpose rather well with those of holo-RXRα LBD. The root mean square deviation (r.m.s.d.) is 1.22 Å for 183 matched Cα's atoms out of 246. Seven helices match rather well (r.m.s.d.: 1.13, 0.88, 0.57, 1.18, 0.67, 0.69, 0.75 Å for H4, H5, H7–H11, respectively). The helices H1, H3, H6 and the β-sheet show larger deviations. The activation helix H12 adopts a conformation similar to that observed in RXRα in the antagonist conformation. However, USP harbors at the same time features characteristic of agonist-bound NR LBDs, namely the length of H11 closer to that of agonist-bound RXRα and the positioning of the phenylalanine residues at its C-terminal part. The coexistence of features related to both agonist and antagonist-bound NR LBD structures is a unique and remarkable property of USP, which will be discussed below in more detail. The helices H1 and H3 contribute to the outermost shell of the LBD. They are less coplanar for USP than for RXRα, the angle between their helical axes being 12.1° larger than the corresponding value in RXRα. This is clearly correlated to the path adopted by the loop L1–3 connecting H1 to H3. It induces a considerable difference in the positioning of the N- and C-terminal parts of H3 compared with agonist and antagonist-bound RXRα LBDs, resulting in a more straight helix, as depicted in Fig. 2. The N-terminal region of H3 (Pro-240 to Cys-250) is displaced outwards the protein core in a substantial manner. It is tilted by about 24° with respect to the same region in holo RXRα. This position is intermediary between the positions of the N-terminal region seen in the apo RXRα and the holo RXRα LBD structures (data not shown). The outward bending of the C terminus of H3 (by about 10°) has repercussions on the positioning of the neighboring loops, L3–4 and L8–9. The loop L3–4, which is part of the signature region of NRs (17Wurtz J.-M. Bourguet W. Renaud J.-P. Vivat V. Chambon P. Moras D. Gronemeyer H. Nat. Struct. Biol. 1996; 3: 87-94Crossref PubMed Scopus (682) Google Scholar), is displaced laterally by about 1.8 Å and bent in the direction of L8–9, which itself is pushed outwards by about 1.5 Å. The conformation adopted by the connecting loop L1–3 is unusual and essential for the stabilization of the actual structure of USP LBD. This contrasts with the observation that this loop usually behaves, in most NRs, as a very flexible region. For hsRXRα, the crystal structures of both apo (18Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Crossref PubMed Scopus (1065) Google Scholar) and holo (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar) conformations show substantial differences in the region connecting helices H1 and H3. In the holo-RXRα LBD structure, L1–3 consists of an extended loop passing above the β-sheet. The apo form exhibits an additional helix in this region, which unfolds in the holo form. In the apo to holo transition, L1–3 also moves substantially. As suggested from the comparison of the apo-RXR and the holo-RXR and RAR LBD structures, L1–3 might act as a molecular spring accompanying the conformational changes which take place upon ligand binding (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar, 22Renaud J.-P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar). For ligand-bound RARγ LBD, the conformation of L1–3 is similar to that of holo-RXRα, except that it contains a C-terminal region forming a so-called Ω-loop (22Renaud J.-P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar). Interestingly, for ER LBDs, L1–3 follows a different path than in the retinoic acid receptors (24Brzozowski A.M. Pike A.C.W. Dauter Z. Hubbard R.E. Bonn T. Engström O. Öhman L. Greene G.L. Gustafsson J.-A. Carlquist M. Nature. 1997; 389: 753-758Crossref PubMed Scopus (2945) Google Scholar, 25Shiau 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 (2252) Google Scholar, 27Pike A.C.W. Brzozowski A.M. Hubbard R.E. Bonn T. Thorsell A.-G. Engström O. Ljunggren J. Gustafsson J.-A. Carlquist M. EMBO J. 1999; 18: 4608-4618Crossref PubMed Scopus (912) Google Scholar). It passes between helix H3 and the β-sheet, tightly packed to the protein core. For the USP LBD, L1–3 adopts none of these conformations. Its path (Val-220 to Pro-239) was unambiguously inferred from electron density maps, as demonstrated in Fig.3 A. Only a few residues at the beginning of the loop, Asp-222, Pro-223, and Ser-224, were included as alanines due to the weak electron density of their side chains. Accordingly, the temperature factors of these residues are higher (60–64 Å (2Lezzi M. Bergman T. Mouillet J.-F. Henrich V.C. Arch. Insect Biochem. Physiol. 1999; 41: 99-106Crossref Scopus (34) Google Scholar)) than those of the other amino acids of L1–3 (36 Å (2Lezzi M. Bergman T. Mouillet J.-F. Henrich V.C. Arch. Insect Biochem. Physiol. 1999; 41: 99-106Crossref Scopus (34) Google Scholar) in average over L1–3). The first residues composing L1–3 form a path that crosses the region of H3 comprising Gln-256 to Val-262. The next residues (Glu-226 to Pro-234) delineate an extended loop that runs along H3. Finally, the last five residues of L1–3 (Asp-235 to Pro-239) form a loop rather similar to the Ω-loop observed in the RARγ LBD (22Renaud J.-P. Rochel N. Ruff M. Vivat V. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 378: 681-689Crossref PubMed Scopus (1030) Google Scholar). L1–3 adopts a rather tensed conformation, which allows it to establish direct contacts with residues in helices H3, H11, and H12 and stabilize them in their actual position (see Fig. 3 B). This is important, because these helices are the structural elements that are shown to undergo the largest conformational changes upon ligand binding (19Egea P.F. Mitschler A. Rochel N. Ruff M. Chambon P. Moras D. EMBO J. 2000; 19: 2592-2601Crossref PubMed Google Scholar). The observed conformation of L1–3 is relevant to the physiological state of the recep

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Crystal Structure of the Ligand-binding Domain of the Ultraspiracle Protein USP, the Ortholog of Retinoid X Receptors in Insects