Binding of Ligands and Activation of Transcription by Nuclear Receptors

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April 14, 2001 17:50 Annual Reviews AR128-14

Annu. Rev. Biophys. Biomol. Struct. 2001. 30:329–59Copyright c© 2001 by Annual Reviews. All rights reserved

BINDING OF LIGANDS AND ACTIVATION OF

TRANSCRIPTION BY NUCLEAR RECEPTORS

Anke C. U. Steinmetz, Jean-Paul Renaud,and Dino MorasLaboratoire de Biologie et Genomique Structurales, CNRS UPR 9004,Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP,BP 163, 67404 Illkirch Cedex, France; e-mail: [email protected],[email protected], [email protected]

Key Words mechanism of activation, isotype specificity, antagonism,partial agonism, conformational change

■ Abstract Nuclear receptors (NRs) form a superfamily of ligand-inducible trans-cription factors composed of several domains. Recent structural studies focused ondomain E, which harbors the ligand-binding site and the ligand-dependent transcriptionactivation function AF-2. Structures of single representatives in an increasing numberof various complexes as well as new structures of further NRs addressed issues suchas discrimination of ligands, superagonism, isotype specificity, and partial agonism.Until today, one unique transcriptionally active form of domain E was determined;however, divergent tertiary structures of apo-forms and transcriptionally inactive formsare known. Thus, recent results link the transformation of NRs upon ligand binding toprinciples of protein folding. Furthermore, the ensemble of NR structures, includingthose of DNA-binding domains, provides one of the foundations for the understandingof interactions with transcription intermediary factors up to the characterization of thelink between NR complexes and the basal transcriptional machinery at the structurallevel.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330RETINOIC ACID STEREO-ISOMERS AND THEIR DIFFERENTIAL

RECOGNITION BY RAR AND RXR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337BINDING OF VITAMIN D TO VDR AND THE STRUCTURAL BASIS

OF SUPERAGONISM OF 20-EPI LIGANDS. . . . . . . . . . . . . . . . . . . . . . . . . . . 339ISOTYPE SPECIFICITY OF RARs AND ERs. . . . . . . . . . . . . . . . . . . . . . . . . . . 341THE MECHANISM OF THE STRUCTURAL TRANSITION FROM

THE TRANSCRIPTIONALLY INACTIVE TO THE ACTIVE FORMOF DOMAIN E AND VICE VERSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

PARTIAL AGONISM AND FULL ANTAGONISM . . . . . . . . . . . . . . . . . . . . . . . . 347

1056-8700/01/0610-0329$14.00 329

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330 STEINMETZ ET AL

USP AND THE UNITING OF STRUCTURAL FEATURES OBSERVEDIN TRANSCRIPTIONALLY ACTIVE AND INACTIVE FORMS . . . . . . . . . . . . 349

HETERODIMERS OF RXRs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351BINDING SITE OF COREPRESSORS AND DISCRIMINATION OF

TRANSCRIPTION INTERMEDIARY FACTORS: RARs, TRs, ANDREV-ERBs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

CONCLUSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

INTRODUCTION

Nuclear receptors (NRs) such as estrogen and retinoic acid receptors (ER and RAR)form a superfamily of ligand-inducible transcription factors directly interactingwith DNA (25). They integrate the signals of a number of different and independentpathways; thus they are involved in the regulation of many processes fundamentalfor pre- and postnatal development of the organism and maintenance of home-ostasis. NRs have been identified in a wide range of metazoa, such that homologsof a single representative can be found in distantly related taxa. For instance, ho-mologs of the retinoic acid receptor related orphan receptors (RORs) are crucialfor diverse functions in chordata (e.g.homo, mus) (1, 26), arthropoda (e.g. crus-taceaHomarus, hexapodaeDrosophila, Manduca, Tenebrio) (16, 40, 46, 70), andpseudocoelomates (e.g.Caenorhabditis) (39). However, to date, no NRs are foundin other eukaryotes as plants and fungi. This permits the locating of the origin ofNRs after the separation of the eukaryotic opisthokonts into the branches leadingto fungi and to metazoa (44).

The superfamily of NRs consists of six phylogenetic families including pro-teins of homologous sequence for which no ligands are known, so-called orphanreceptors (41, 52). Isoforms and subtypes are known for most NRs; those char-acterized in more detail differ in selectivity of ligands, transcriptional activity,and distribution in tissues. The known natural ligands of NRs arise from biosyn-thetic pathways that originate from isoprenoid moieties and involve enzymes ofthe P450 superfamily (50, 56; Figure 1). One key intermediate is all-trans-retinalfor retinoic acid derivatives, which are the ligands of retinoic acid receptor (RAR)and retinoid receptor (RXR). Other key intermediates are squalene, cholesterol,and pregnenolone for vitamin D [vitamin D receptor (VDR)], corticoids [glucocor-ticoid and mineralocorticoid receptors (GR and MR)], and reproductive steroids[estrogen, progesterone, androgen receptors (ER, PR, and AR)]. Thyroid hormones[thyroid hormone receptor (TR)], leukotrienes, and prostaglandins that bind andactivate the peroxysome proliferator-activated receptors (PPARs) represent excep-tions at the present status of knowledge. However, the chemical and biosyntheticrelationships of the ligands do not correlate consistently with the phylogeneticrelationships of the families to which their respective receptors belong.

Endogenous ligands of pregnane X receptor (PXR), constitutive androstane re-ceptors (CARs), liver X receptors (LXRs), and farnesol X receptor (FXR) were

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HOW NUCLEAR RECEPTORS WORK 331

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332 STEINMETZ ET AL

identified as steroids, cholesterol derivatives, and bile acids. These receptors regu-late the catabolism of steroids and the metabolism of cholesterol and its derivativesby the control of transcription of genes of the P450 superfamily (22, 36). Further-more, the P450 enzymes of families IIB and IIIA, the biosynthesis of which iscontrolled by PXR and CARs, metabolize xenobiotics. Some of these xenobioticsare exogenous agonists of PXR and CARs such that they activate the metabolicpathways of their own catabolism. The complexity of transcriptional regulationof P450 genes by NRs, control of NRs by steroids, and steroid metabolism viaP450 enzymes suggests that the diversification of NRs evolved in parallel withthe evolution of P450 enzymes constituting the biosynthetic pathways of newlyemerging ligands.

Classical steroid NRs interact transiently with chaperones that appear to assistin their folding or help their refolding (57, 74). Based on studies with GR, it wasproposed that the dynamic assembly of heterocomplexes of steroid NRs with highmolecular weight immunophilins and chaperones rapidly transports steroid NRsalong the cytoplasmic movement machinery (58).

NRs repress or activate transcription by participating in the constitution of large,multimeric protein complexes on the promoter of their target genes (23). The func-tional state of NRs is changed upon the binding of ligand such that corepressorsare released and coactivators are recruited. Corepressors such as nuclear receptorcorepressor (NCoR) interface between apo-NRs, Sin3, and histone acetylase com-plexes, which harbor histone deacetylase activity thus condensing the nucleosome.At least two types of complexes of NRs activated by agonists and coactivators ex-ist. They possibly form one after the other, both being anchored by NRs on thepromoter. Complexes of type I are assembled by coactivators of the p160 familythat assist in recruiting the histone acetylase activity of complexes of CREB-binding protein/p300 (CBP/p300) and p300/CBP associated factor (p/CAF) to thepromoter in addition to their own histone acetyl transferase activity. This processis correlated with the local decondensation of chromatin. Complexes of type IIare formed via coactivators of VDR interacting proteins (DRIP)/TR associatedprotein (TRAP) type that bridge NRs to mediator/SRB subunits associating withRNA polymerase II holoenzyme. Acetylation of the p160 coactivators by CBP isproposed to trigger the dissociation of the complex of type I and allow for theformation of the complex of type II. Recent data suggest that the degradation ofselected components of the multimeric complexes by the ubiquitin proteasomepathway further assists the exchange of coactivators (38, 42). Thus, the changingconstitution of the complexes repressing or activating transcription appears to re-sult in at least one chain of events that leads from decondensation of chromatin byacetylation to subsequent transcription by RNA polymerase II holoenzyme.

Direct interactions of NRs with basal transcription factors were determined(30, 35, 65). Thus, ER and PR interact with transcription factor IIB (TFIIB) andsubunits of the TFIID complex. Furthermore, ER was shown to associate with theTATA box binding protein (TBP); RXR and TR were reported to interact with asubunit of the TFIID complex, and VDR associates with TFIIB. The formation of

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these complexes was not modulated by the presence of ligands as far as tested.Furthermore, direct interactions of NCoR with TFIIB and subunits of the TFIIDcomplex were reported (47). However, at the moment neither the temporal northe spatial context of these interactions are clearly defined for the course of theactivation of transcription.

NRs are composed of five to six domains (25, Figure 2). Domain C is theDNA-binding domain that recognizes the so-called ligand response element in thepromoter region of the target gene via two zinc-finger modules. Domain E, whichharbors the ligand-binding site and a ligand-dependent transcription activationfunction (AF-2), is located C-terminally. Domain D is a linker between domains Cand E and appears structurally flexible. Sequence and length of domain D varyconsiderably between different receptors. Regions interacting with heat shockproteins and harboring the nuclear localization signal are identified at the transitionof domains C and D and in domain E. The N-terminal domain A/B harboringan autonomous transcription activation function called AF-1 as well as the C-terminal domain F, the function of which is not yet well elucidated, are highlydivergent if at all present. Thus, a minimal NR is constituted of domains C, D,

Figure 2 Scheme of the modular structure of NRs. The N-terminal domain A/B harbors theautonomous transcription activation function AF-1. Domain C achieves binding to DNA via twozinc-finger modules; domain D is a flexible hinge region. Domain E harbors the ligand-binding siteand the ligand-dependent transcription activation function AF-2. Domain F exists only in someNRs. Tertiary structures of domain C and domain E of representative NRs were determined byNMR and crystallography. The tertiary structures of domains A/B, D, and F are not known norare the relative positions of domains A to F with respect to each other.

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334 STEINMETZ ET AL

and E. The predominant signal regulating the transcriptional activity of NRs is thebinding of a ligand. However, the transcriptional activity of ligand-free and ligand-bound receptors can be fine-tuned by phosphorylation as the result of a cascade ofphosphorylation events that signals the stimulation of cell-surface receptors to thenucleus (25). Furthermore, synergy observed between the transcription activationfunctions AF-1 and AF-2 of ERα and PPARγ , for example, seems to be mediatedby coactivators such as TIF2 and p300 (3, 21, 37).

The crystallographic structures of domain E of NRs belonging to families 1, 2,and 3 were determined either in the apo-form or in complex with various endoge-nous ligands, synthetic agonists, and antagonists (60). Domain E is composed ofan antiparallelα-helical sandwich with the insertion of a smallβ-sheet (Figure 3).All structures of domain E of NRs in complex with ligands determined so far showthe ligand-binding site between theβ-sheet, helix 3, helix 5, helix 11, and helix 12.The analysis of these structures reveals that helix 1, the C-terminal part of helix 3to helix 5, and helices 7 to 10 represent the scaffold common to all structures ofwhich the backbone superimposes well independent of the phylogenetic relation-ship (72). The apo-receptor undergoes conformational changes upon the bindingof the agonist, which dissociates the complex with corepressors and permits theformation of a complex with coactivators. Helix 5, theβ-sheet, helix 6, and helix 10

Figure 3 Scheme of the topology representing the antiparallelα-helical sandwich fold ofdomain E of NRs.α-helices are indicated as rectangles labeled H1 to H12. The strands oftheβ-sheet are indicated as arrows and labeled s1 and s2.

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are slightly shifted upon activation. Helix 11, helix 12, and loop 11–12, as wellas the segment between helix 1 and helix 3, undergo major rearrangements uponligand-binding (Figure 4). The greater the movements of a structural element uponactivation, the greater the structural variations observed between different recep-tors in these regions and different complexes of the same receptor. The structuraltransition of the transcriptionally inactive and active forms, called “mouse-trap”mechanism and inferred from the first comparison of domain E of RXR in itsapo-form with domain E of RAR in complex with the agonist all-trans retinoicacid (61), involves the placement of helix 12, which is the core of the ligand-dependent AF-2 against the core of the protein in order to close the entranceof the ligand-binding pocket and generate the binding site of the LXXLL motifof coactivators (11, 51, 66). In order to achieve this placement of helix 12, ma-jor rearrangements of helix 11, loop 11–12, and the connection between helix 1and helix 3 occur.

The ternary complexes of a coactivator fragment, an agonist, and either PPAR,TR, or ER reveal the localization and binding-mode of the LXXLL motif. TheLXXLL motif is part of anα-helical stretch, which packs with hydrophobic sidechains against the core of the receptor in a shallow hydrophobic groove betweenresidues located in helices 3, 4, and 12 (Figures 4b and 4c). The motif is heldin place via electrostatic interactions with charged side chains of a lysine residuelocated at the C-terminus of helix 3 and a glutamate residue located in helix 12that cap either end of the helical segment of the coactivator. This arrangementof charged side chains that anchor the LXXLL motif was termed “charge clamp.”The structures of domain E of ERα in complex with selective ER modulators showthat helix 12 can bind with the core of the AF-2 in precisely the same locationas the LXXLL motif of the coactivator fragments (Figures 4c and 4d) (8, 66).The structure of domain E of ERβ in complex with 4-hydroxytamoxifen suggeststhat helix 12 can bind in the same location; however, the extremely high thermalfactors raise doubts about the stability of this conformation or the precise location ofhelix 12 (55).

Recent structural investigations of NRs focused on domain E. An importantnumber of crystallographic studies centered on the mode of ligand binding. Thus,the structures of domain E of RARs in complex with various agonists and an-tagonists revealed the basis of isotype selectivity of these ligands. The isotypespecificity of ERs was addressed by the determination of domain E of ERβ incomplex with genistein and 4-hydroxytamoxifen (55). The determination of do-main E of RXRα in complex with 9-cis retinoic acid elucidated the differentialbinding of this ligand to RARs and RXRs as well as the discrimination of 9-cisand all-trans retinoic acid by RXRs (15). The structure of domain E of VDRin complex with vitamin D3 and synthetic ligands was determined in order todefine the structural basis of the so-called superagonist activity of 20-epi lig-ands (63, 67). The mechanism of activation of domain E upon binding of ago-nists to NRs was addressed by experimental and theoretical approaches. Thus, theinitially proposed mouse-trap mechanism could be refined by crystallography on

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the basis of the complex 9-cis retinoic acid/RXRα. This complex suggests thatentropic contributions strongly determine the distribution of numerous coexistingstructural states (15). The structure of domain E of a lepidopteran USP determinedby crystallography presents structural features of both transcriptionally active andinactive conformations so far unique to lepidopteran USPs. This conformationis most likely induced by a copurified phospholipid (4). The structure indicatesan entrance to the ligand-binding site and underlines the polymorphism of NRs.The NMR spectroscopic study of PPARγ in absence and presence of the agonistrosiglitazone underlines that binding of agonists stabilizes a distinct form of do-main E, which is selected out of a large ensemble of conformational states (29). Asimulation of the escape of all-trans retinoic acid from its binding-site in RARγsuggests that ligands bind to an open form of domain E, which is already close toits transcriptionally active form (5).

A particular aspect of the binding of agonists to NRs is that only one tran-scriptionally active form of domain E of NRs is established. The crystal structuresof VDR in complex with 20-epi ligands suggest that the superagonistic activityof these ligands is based on their high affinity to the receptor and not on theinduction of a different, transcriptionally more active form of the receptor (67).Furthermore, the crystal structure of the heterodimer of agonist-bound domains Eof RXRα and PPARγ suggests that so-called permissive signaling via RXRs inheterodimers formed with, for example, PPARs and FXR is based on the stabiliza-tion of the canonical transcriptionally active form of the heterodimeric partner dueto a particular salt bridge (19). However, multiple apo- and antagonist-forms weredetermined by crystallography, indicating that coexisting conformations are ener-getically not very different. Thus, the structural polymorphism of NRs suggestedby the NMR spectroscopic experiments carried out with PPARγ finds expressionfor example in the diverse conformations of domain E of ERs upon binding ofligands. It was already noted that the N- and C-terminus of helix 12 of ERα are notalways formed by the same residues, and that the segment connecting helices 1 and3 and loop 6–7 assume different conformations in structures of ERα in complexwith 4-hydroxytamoxifen and raloxifene (66). The structures of domain E of atriple cysteine-to-serine mutant of ERα in complex with estradiol and of ERβ incomplex with genistein present further possible conformations of ERs (20, 55).The structures of domain E of PPARs determined by crystallography depict an-other ensemble of distinct conformations. The crystal structure of the heterodimerof domains E of RARγ in complex with the antagonist BMS614 and of the mutantRXRαF318A in complex with oleic acid reveals that the particular mechanism ofantagonism based on the repositioning of helix 12 in the coactivator binding-siteis not unique to ERs (7).

The biochemical characterization of partial agonists of ERs, RXRs, and PPARsby affinity measurements and transient transfection assays, in combination withthe crystallographic determination of the corresponding domain E complexes, re-veals that partial agonism is based at the molecular level on the coexistence of

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transcriptionally active and inactive forms of domain E while bound to the lig-and (Figure 4). This implies that nonproductive binding exists, and that one basisof partial agonism is the insufficient stabilization of helix 12 in the transcrip-tionally active form of domain E. Examples are the complexes GW0072/PPARγ ,genistein/ERβ, oleic acid/RXRαF318A, and estradiol/triple cystein-to-serine mu-tant of ERα. Thus, the picture evolves to ligands tipping the scales of the equi-librium of coexisting conformations of NRs as it can be described by statisticalthermodynamics (17). The shift of the conformational equilibrium can be assistedby the presence and concentration of transcription intermediary factors as estab-lished for ERs and fragments of coactivators. Furthermore, biochemical studiessupported by homology modeling suggest that the binding sites of coactivators andcorepressors partially overlap and suggest a mechanism for the exchange of tran-scription intermediary factors upon binding of agonists (49, 54, 59). These resultsfurnish the first explanations at the structural level regarding how the biologicaland biochemical contexts play important roles for the transcriptional activity ofNRs. The recent results in the structural biology of NRs link the mechanisms un-derlying the structural transformation of NRs upon ligand binding to mechanismsof protein folding. The crystallographic structures of domain E of NRs, as well asthe crystallographic and NMR structures of domain C, are a solid basis for dynam-ical studies addressing specific aspects of the structural transition of NRs uponbinding of ligands by methods such as fluorescence resonance energy transfer andelectron paramagnetic resonance. Furthermore, these structures serve as a start-ing point to address the structural characterization of multimeric complexes withtranscriptional intermediary factors. They also provide a basis for the structuraldetermination of the link between NRs complexes and the basal transcriptional ma-chinery, employing all methods of structural biology such as electron microscopy,crystallography, and atomic force microscopy.

RETINOIC ACID STEREO-ISOMERS AND THEIRDIFFERENTIAL RECOGNITION BY RAR AND RXR

Retinoic acid derivatives regulate the transcriptional activity of RARs and RXRs,which control complex gene networks during pre- and postnatal development andhomeostasis of the adult organism (31, 48). The principal functional unit respond-ing to the retinoid signal is a heterodimer composed of RAR and RXR. However,RXRs activate transcription in heterodimeric complexes with many other NRssuch as TRs, VDR, or PPARs. RXRs bind and respond only to 9-cis retinoic acid,whereas RARs are activated by all-trans and 9-cis retinoic acid (Figure 5). Theaffinity of 9-cis retinoic acid to RXRs is about one order of magnitude lower thanits affinity to RARs.

The comparison of the crystallographic structures of domain E of RARγ incomplex with either all-trans or 9-cis retinoic acid revealed that both ligands

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induce a perfectly identical transcriptionally active form of the protein (34). Theligands fit in the binding site of the receptor tightly. They are forced to adjust tothe protein such that the prerequisite for their binding is their malleability: 9-cisretinoic acid is less kinked when bound to the protein than in the lowest energyconformation of this compound, and all-trans retinoic acid is more bent in thecomplex than in the lowest energy conformation.

Domain E of RXRα in complex with 9-cis retinoic acid assumes the transcrip-tionally active form (15, 19). However, the ligand adopts a sharper bend and isslightly shifted and rotated around its aliphatic polyunsaturated chain with respectto the rigid scaffold of domain E of RXRα in comparison to 9-cis retinoic acid incomplex with domain E of RARγ . Furthermore, theβ-ionone ring is differentlyoriented with respect to the aliphatic chain of 9-cis retinoic acid upon binding toRXRα and RARγ (Figure 6).

In conclusion, both, RARγ and RXRα impose energetically less favorableconformations on their ligands as compared to their ideal conformations in solution.The shape of the ligand-binding site of RARs permits binding of both all-transor9-cis retinoic acid owing to a certain malleability of these compounds. In contrast,the more pronounced bend of the ligand-binding site of RXRs discriminates all-transand 9-cis retinoic acid because of the basic rigidity of the conjugated systemof the aliphatic polyunsaturated chain.

BINDING OF VITAMIN D TO VDR AND THESTRUCTURAL BASIS OF SUPERAGONISMOF 20-EPI LIGANDS

VDR controls transcription as a heterodimer with RXR bound to the cognate hor-mone response element in the promoter region of its target genes. The principal en-dogenous agonist of VDR participating in the regulation of calcium and phosphatemetabolism, inducing cell differentiation, and having immunosuppressive effectsis 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (27; Figure 5). Thus, 1,25(OH)2D3and its analogs are indicated in a wide range of therapies. However, the therapeuticapplication of natural vitamin Ds is limited because their effects on homeostasisand cell differentiation are not well separated. Differentially acting agonists ofVDR with reduced side effects have been developed (6). The differences of theclinical profiles, which are based on differential effects on the transcription oftarget genes, are anchored in domain E and its AF-2 because VDR does not con-tain an AF-1. One group of synthetic agonists of VDR, called superagonist 20-epiligands, binds with an affinity similar to that of 1,25(OH)2D3. However, they in-duce transcription at concentrations 100-fold below the concentration required for1,25(OH)2D3. Furthermore, they strongly induce cell differentiation, whereas theireffects on calcium metabolism are reduced compared to 1,25(OH)2D3. The struc-tures of domain E of VDR in complex with 1,25(OH)2D3 and two superagonist

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Figure 6 Comparison of the conformations of 9-cis retinoic acid bound either to RARγor RXRα (Brookhaven entry codes 3lbd and 1fby, respectively). 9-cis retinoic acid of thecomplex with RARγ is light gray; 9-cis retinoic acid of the complex with RXRα is darkgray. The ligands were superimposed manually (left). The more pronounced bend of 9-cisretinoic acid when bound to RXRα (dark gray) is evinced by this superposition (left). Theproteins were superimposed by a least-squares fit and subsequently deleted from the model(right). The different orientations and locations of theβ-ionone ring and the shift of thecarboxyl group with respect to the protein core are evinced by the superposition (right).

20-epi ligands reveal their mode of binding and the structural basis for their dis-tinct properties with respect to transcription (Figure 5; 63; G Tocchini-Valentini,N Rochel, JM Wurtz, A Mitschler, D Moras, manuscript submitted).

Domain E of VDR in complex with 1,25(OH)2D3 presents the canonical trans-criptionally active form (63). The domain contains an insertion of about 50 residuesin the segment connecting helices 1 and 3. The removal of this insertion doesnot significantly affect ligand binding, transactivation, or heterodimerization withRXRs. The connection between helix 1 and helix 3 is well ordered such that he-lix 2, an additional helix 3n, and the loop regions are observed. This connectionis located in a manner similar to the one observed in ERs. The position of thisconnection and the conformation of the tip of theβ-sheet adapt to each other,and the relative position of helix 6 and loop 6–7 is shifted with respect to therigid scaffold of domain E in comparison to the structures of other NRs. Thesubstituent at C17 of the steroid is located in this region. Thus, the 25-hydroxylgroup forms hydrogen bonds to His-305 and His-397, which are located in loop6–7 and helix 11, respectively. Rings C and D form mostly van der Waals con-tacts in the central region of the ligand-binding site. The conjugated triene of theseco ring B fits tightly in a channel between residues located in loop 5-β, strand1 of theβ-sheet, and helix 3. The C6–C7 bond is observed ins-transconfor-mation. The hydroxyl groups at ring A of 1,25(OH)2D3 form hydrogen bonds toresidues in helix 3 and helix 5. The ligand stabilizes the conformation of helix12 indirectly by interacting with Val-234 in helix 3, Ile-268 in helix 5, and His-397 and Tyr-401 in helix 11. These residues are in direct contact with helix 12.

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Rings A, C, and D of vitamin D adopt the same low energy conformations inthe free state and in the complex with VDR. However, the triene of seco ring Bdeviates by 30◦ from planarity in the complex with domain E of VDR, which isnot observed in the crystallographic structure of free 1,25(OH)2D3. Thus, simi-larly to RARs, VDR locally imposes an energetically elevated conformation onthe ligand.

The two superagonist 20-epi ligands, KH1060 and MC1288, induce exactly thesame transcriptionally active conformation of domain E of VDR as 1,25(OH)2D3does (67). The increased stability of the complexes of VDR with MC1288 andKH1060 is achieved either by an energetically lower conformation of the ligand oradditional interactions between the ligand and the protein. The increased affinityof the 20-epi superagonists to VDR causes their elevated capacity to activatetranscription. The structures demonstrate that a minimum of malleability in theregion of the seco ring B is a prerequisite for the binding of seco steroids to VDR.They further reveal how the nature and flexibility of substituents at C17 and theirpotential to form contacts in the ligand-binding site affect the affinity of a ligand.

ISOTYPE SPECIFICITY OF RARs AND ERs

Isotypes of many NRs are identified (25). Differences in sequence accumulate inthe N-terminal A/B region, including different length and N-termini due to differentpromoter usage and RNA-splicing. Furthermore, the sequences of isotypes differstrongly in domain D. Mutations to various degrees are also observed in domain E.Isotypes often show different tissue distribution and differential expression duringthe development of an organism that serves as the rational basis for the design ofisotype-specific ligands as drugs. For instance, the isotypesα,β, andγ of RAR areimplicated in different diseases, thus representing distinct pharmacological targets(48). The application of retinoids in the treatment of acute promyelocytic leukemiais based on the effect of RARα on differentiation of leukocytes. RARβ representsa possible target in the treatment of different cancers, in particular breast cancer.Retinoids are applied to treat skin diseases such as psoriasis and acne due to theinvolvement of RARγ in these conditions.

The crystallographic analysis of domain E of RARγ in complex with variousisotype selective ligands defines principles for the design of ligands that act ei-ther as a panagonist,β,γ -specific, orγ -selective (33, 7; Figure 5). Panagonistsrequire a minimum of flexibility such that they can adapt to the ligand-binding siteas deduced by the comparison of the complexes of domain E with 9-cis retinoicacid, all-trans retinoic acid, and BMS181156. Furthermore steric interference ofthe ligand at the binding site of theβ-ionone ring with Ser-234 of RARα, whichis an alanine in RARβ and RARγ , has to be avoided. Replacing theβ-iononering of retinoic acid by a sufficiently bulky moiety with concomitant shorteningof the linker to the carboxyl group as in CD564 disfavors binding to RARα.

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Introducing a hydroxyl group at the position equivalent to the 19-methyl groupof retinoic acid generatesγ -selectivity in the case of the R-enantiomers. In thecase of the S-enantiomer BMS270395, the compound may bind to RARγ as ob-served crystallographically, even in the transcriptionally active form of domain E;however, it does not induce transcriptional activity in transient transfection as-says (32). The S-enantiomer assumes a twisted high energy conformation in thecrystallized complex. The crystallization conditions probably stabilize this highenergy ligand-protein complex, which is not detected in transient transfection as-says.γ -selectivity is based on a hydrogen bond formed by the hydroxyl groupto the sulfur atom of Met-272. Met-272 is replaced by an isoleucine residue inRARα and RARβ. Replacing the hydroxyl group by a keto function transformsthe ligand either into a panagonist or aβ,γ -selective agonist depending on po-tential interference with Ser-232 of RARα. The binding-site of RARs fits tightlythe ligands, such that conformational shifts of side chains induced by a particularligand force the side chains of residues not directly in contact with the ligand toadapt. However, the crystallographic structures superimpose very well, except forthese minor adjustments of side chains, and helix 12 is always in the perfect agonistposition.

The crystal structure of the heterodimer of domains E of RARα in complexwith the antagonist BMS614 and the mutant F318A of RXRα demonstrates howselectivity of a ligand to RARα is achieved. Theα-selective antagonist bindswith higher affinity to RARα than to RARβ and RARγ owing to the polar in-teraction between the hydroxyl group of Ser-232 of RARα and the aromatic ringE of the antagonist. This interaction is lost in isotypesβ and γ because Ser-232 in RARα is replaced by an alanine in RARβ and RARγ . The affinity ofBMS614 to RARγ is further diminished owing to steric interference of the com-pound with Met-272 of RARγ , which is an isoleucine in RARα. The loss of avan der Waals contact due to the exchange of Val-395 of RARα against an alanineresidue in RARγ contributes in addition to the reduced affinity of BMS614 toRARγ .

The isotype specificity of ERs requires more complex explanations. Estro-genic agonists, antagonists, and selective ER modulators are applied for instancein the treatments of cancer, female reproductive control, and hormonal replace-ment during menopause (12, 43). Their effects mediated by ERs are extensivelyinvestigated; the mechanisms of their extra-nuclear effects on signaling are lessunderstood (62). However, the differences of the biological roles between the twoisotypes of ERs are not yet well defined (14). Furthermore, the differential affinityof agonists to ERα and ERβ, and their competition with either pure antagonistsor selective ER modulators for either ERα or ERβ do not correlate consistentlywith activation of reporter gene transcription in transient transfection assays byeither isotype (2, 24). The potency of estradiol to activate reporter gene expressionthrough ERβ is slightly reduced compared to its potency of transcriptional acti-vation through ERα. This difference is even more pronounced for the responsesof ERs to genistein, although genistein binds fourfold more tightly to ERβ than

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to ERα (Figure 5). This observation is intriguing because the residues lining theligand-binding sites of ERα and ERβ are conserved except for two conservativemutations.

The crystal structure of domain E of ERβ in complex with genistein revealsthat the ligand binds in a manner similar to estradiol in complex with domain Eof ERα (55). However, helix 12 is positioned in the coactivator binding groovesimilarly to the structures of domain E of ERα in complex with either ralox-ifene or 4-hydroxytamoxifen (Figures 4c, 4d, 4e, and Figure 5). Neverless, genis-tein does not stabilize the same transcriptionally inactive form in ERβ, which4-hydroxytamoxifen and raloxifene induce in ERα. Helix 12 is longer and its axisis tilted by 25◦ in ERβ in complex with genistein as compared to the position ofhelix 12 in the complexes of ERα with raloxifene and 4-hydroxtamoxifen. Theposition of helix 12 in the binding-site of coactivators explains the reduced potencyof genistein to activate transcription of reporter genes via ERβ. The mutation ofAsn-348 of ERα to a lysine residue in ERβ is proposed to destabilize helix 12 inthe transcriptionally active form and induce the repositioning of helix 12 in thecoactivator binding site.

THE MECHANISM OF THE STRUCTURAL TRANSITIONFROM THE TRANSCRIPTIONALLY INACTIVE TO THEACTIVE FORM OF DOMAIN E AND VICE VERSA

The structural transformation of domain E upon binding of agonists to NRs wasaddressed recently by crystallography, a theoretical approach, and NMR. Theproposal of the “mousetrap” mechanism was based on the comparison of thecrystal structures of domain E of RXRα in the apo-form and domain E of RARγin complex with all-trans retinoic acid (61; Figures 4a and 4b). The assumptionthat the binary complex of RARγ represents the transcriptionally active form ofdomain E of NRs is supported by several other structures of domain E of NRs incomplex with strong agonists. Some of these complexes contain a small fragment ofcoactivators indicating the binding site and the mode of interaction of coactivators(Figure 4c). All complexes indicate that the ligand-binding site is located in aregion that presents a very different topology in domain E of apo-RXRα, wherethe cavity is not properly formed and partly obstructed. Thus, several questionsimpose: Do RXRs indeed have the same ligand-binding site? Does a differentbinding site and a different structural conformation upon ligand binding accountfor the ability of RXRs to serve as heterodimeric partner? Does the apo-form ofRXRα represent a general apo-form of NRs? The relevance of the latter question issharpened by the fact that domain E of PPARs crystallized in the ligand-free statein a tertiary structure with helix 12 already located close to the position observedin the transcriptionally active complexes of domain E of PPARs with agonists(51, 68).

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The crystal structure of domain E of RXRα, in complex with 9-cisretinoic acid,was determined independently, once as a monomer and once as the heterodimericpartner of domain E of PPARγ in complex with the agonist rosiglitazone andpeptides derived from the coactivator SRC-1 (15, 19; Figure 5). Both structuresreveal that the ligand-binding site of RXRs is located in the same region as inother NRs. They validate the “mousetrap” mechanism in the presence and absenceof a heterodimerizing partner because domain E is observed in both structuresin the canonical transcriptionally active form. Furthermore, the fragment of thecoactivator SRC-1 is located in the coactivator binding sites of both PPARγ andRXRα as established previously in PPAR, TR, and ER. The comparison of theapo- and holo-forms of domain E of RXRα strongly suggests that the structuralelements assuming different tertiary arrangements are flexible in the apo-formbecause helix 11 has to first swing out of the binding-site in order to allow theligand to enter afterwards. Thus, the transformation of the apo- to the holo-formof domain E of RXRα takes place on the rigid scaffold formed by helix 1, theC-terminal part of helix 3 to helix 5, and helices 7 to 10. Theβ-sheet and he-lix 6 adapt by slightly shifting. Helix 11 aligns in the continuity of helix 10,and helix 12 repositions the core of the AF-2 in the transcriptionally active formof the domain. The N-terminus of helix 3 kinks such that—with the concomi-tant rearrangement of helix 12—the ligand is entirely closed off from the solvent(15; Figure 7).

The structures indicate that the equilibrium in solution between the crystallo-graphic apo-form (NR blocked), a structure accessible to ligands (NR open), andthe transcriptionally active form (NR active) is strongly influenced by entropiccontributions (15; Figure 8). The three large hydrophobic side chains of Leu-441,Phe-437, and Phe-438 located on helix 11 obstruct a part of the ligand-bindingsite. In this position, they are buried in the interior of the protein. In the transcrip-tionally active form, they are exposed on the surface of domain E. This exposureof hydrophobic side chains is only partly compensated for by arranging the sidechains of Leu-436 and Phe-439 on helix 11 in the interior of the protein such thatthey line the ligand-binding site in the transcriptionally active form of domain E.

Comparing the structures of domain E of RXRα in complex with 9-cis retinoicacid and of domain E of RARγ in complex with various strong agonists revealsthat the relative position of helix 6 and theβ-sheet, with respect to the rigid scaf-fold, are distinct in RXRα and RARγ . The adaptations of helix 6 and theβ-sheetupon binding of 9-cis retinoic acid to RXRα are small. Furthermore, theÄ-loopof the segment connecting helices 1 and 3 in RARγ partly envelops helix 6, whichis not observed in the structure of domain E of RXRα in complex with 9-cisretinoic acid. Enhanced molecular dynamics simulation of the escape of all-transretinoic acid from domain E of RARγ was applied in order to obtain an unbi-ased picture of the cohesive strength of the structural elements of domain E ofRARγ and to gain insights into the structural transformations accompanying exitand entrance of the ligand (5). The simulation shows that helix 12 readily movesaway from the ligand-binding site, thus opening the gate for the exit of the ligand

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Figure 8 The equilibrium of transcriptionally relevant forms of NRs. The central equi-librium defined by the constant Kd1 exists between the receptor accessible to ligand andthe binary receptor-ligand complex (NR open and NR open∗ L, respectively). The tertiarystructures of domain E of NRs probably assume a wide range of conformations in the ab-sence of ligand, which are energetically not very different. They may transiently assumethe transcriptionally active and inactive forms observed by crystallography. However, in theabsence of ligands, these forms are not stable. An agonist stabilizes the transcriptionallyactive form of the binary complex (NR active∗ L), which permits the formation of signifi-cant amounts of the ternary complex by binding of coactivators. An antagonist stabilizes atranscriptionally inactive form of the binary complex (NR inactive∗ L). Partial agonists donot sufficiently stabilize the transcriptionally active conformation and permit the formationof transcriptionally inactive conformations. In solution, an equilibrium between the trans-criptionally active and inactive forms exists. The crystal structure of domain E of RXRα es-tablishes that this receptor may assume a conformation that is not accessible to ligands (NRblocked). This implies that the structural elements involved in the conformational changeare flexible in solution and an equilibrium between NR blocked and NR open exists. Thisparticular apo-conformation is probably not accessible to all NRs.

(Figure 9a). Kinked helix 3 straightens, thus further opening the exit channel. Inspite of the straightening of helix 3, theÄ-loop remains initially close to helix6. In this manner, the channel for the exit of the ligand is confined to the regionbetween helix 11 and the N-terminus of helix 3. Three paths for the transition fromthe crystal structure of domain E of RARγ to a structure modeled in analogy to theapo-structure of RXRα were calculated by the conjugate peak refinement method

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(5; Figure 9b). These calculations suggest that the conformational space of domainE of RARs, in absence of ligand, does not contain large energy minima funnelingthe protein to one particular conformation in the apo-form. Inversely, these calcu-lations and the enhanced molecular dynamics simulation suggest that the ligandbinds to an open form of the domain, which is already close to the transcrip-tionally active form. Once bound, the ligand stabilizes the transcriptionally activeform.

PPARs represent targets in the treatment of diabetes and cardiovascular dis-eases related to obesity because they are involved in the regulation of lipid andglucose metabolism. They participate in the control of genes that encode proteinsresponsible of metabolism and storage of fatty acids. Eicosanoid derivatives andfatty acids activate transcription of reporter genes via PPARs. The crystal struc-tures of domain E of PPARs in complex with various agonists reveal the proteinin the canonical transcriptionally active form in which the position of helix 12 isstabilized via interactions with the acidic head groups of these ligands (51, 73).

The study of the transformation of domain E of PPARγ upon binding of lig-and by NMR provides experimental evidence for the existence of a large en-semble of multiple conformations prior to the binding of ligand (29). The spec-tra of domain E of PPARγ indicate that up to about half of the structure ismobile before the addition of the agonist rosiglitazone. The regions, which didnot give rise to signal, cluster around the ligand-binding pocket and include thebinding site of coactivators. Addition of rosiglitazone increased the signal suchthat the structure was reconstructed in the transcriptionally active form as deter-mined by crystallography. Thus, the binding of agonist restricts a large conforma-tional space of the apo-receptor such that the entire binding site of coactivators isconstituted.

Several structures of PPARs, which had been crystallized without the additionof ligands, have been reported (51, 68). In the homodimer of apo-PPAR, helix 12is located in one subunit close to the canonical position observed in the transcrip-tionally active form and in the other such that it binds in the coactivator bindingsite of a crystallographically related molecule (51). In apo-PPAR crystallized as amonomer, helix 12 is located close to the position observed in the transcriptionallyactive form (68). Thus, the crystallographic structures underline the coexistence ofdistinct conformations prior to the binding of agonists. Furthermore, PPARs havea rather large ligand-binding site that is not entirely closed off from the solvent inthe reported apo-forms nor after the binding of agonists. The reported apo-formscould correspond to open forms that permit the access of ligand to its binding sitebefore stabilization of the transcriptionally active form. Alternatively, a copurifiedfortuitous ligand, such as a fatty acid, could have stabilized the structure close tothe transcriptionally active conformation. Weak binding of the ligand, combinedwith the influence of crystal contacts, may have permitted the particular asymmet-ric assembly in the crystal. Disorder and low occupancy of the fortuitous ligandmight have hidden the presence of the copurified molecule.

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PARTIAL AGONISM AND FULL ANTAGONISM

The issue of partial agonism versus full antagonism is of predominant importancein the treatment of breast cancer by antiestrogens (12, 43). Partial agonism ofantiestrogens describes the potential of ligands to elicit responses that correspond tothe effects of estrogens in some tissues, but inhibit the effects of estrogens in others(43). The mechanisms underlying partial agonism of antiestrogens are complex androoted in different aspects of estrogenic biology. The biochemical determination ofaffinity, recruitment of transcription intermediary cofactors, and potency to activatetranscription in transient transfection assays characterizes partial agonism of aparticular ligand as the discrepancy of these parameters in comparison to a givenstandard (e.g. the response of estradiol elicited via ERα). The concept of partialagonism was transferred to, for instance, RXRs and PPARs in this biochemicalsense.

The structure of domain E of ERβ in complex with genistein evinces thatone mechanism underlying partial agonism is based on a reduced stabilizationof the transcriptionally active form such that an equilibrium between ligand-associated transcriptionally inactive and active forms of domain E is established(55; Figure 4e). Thus, the crystal structure of domain E of ERβ in complex withgenistein underlines that a strongly binding ligand with the potential to activatetranscription via AF-2 does not necessarily stabilize the transcriptionally activeform of NRs in correlation with its affinity. Considering the significantly differentaffinities of ERα and ERβ to genistein and the very similar ligand-binding sites ofthe isoforms with a presumably conserved binding mode of genistein in both iso-types, further suggests that both affinity and potency of a ligand cannot be reducedsolely to the number and strength of direct contacts within the ligand-bindingsite. Thus, already on the structural level, the entire functional mechanism of thereceptor contributes to the distinct isotype specific responses. In such cases, theintracellular environment will determine how the ligand will effect the homeostaticequilibrium of the cell.

The biochemical and crystallographic characterization of a triple cysteine-to-serine mutant of ERα compared to wild-type ERα resembles the comparison ofisotype specific responses of ERs (20). The triple cysteine-to-serine mutant of ERα

(C318S, C417S, C530S) binds estradiol with the same affinity as the wild-typereceptor, and the transcriptional activity induced by estradiol is entirely abolishedby full antagonists. However, the maximal transcriptional activity of the triple mu-tant is limited to about half of the wild-type activity. The effects of the mutationson the transcriptional activity of the receptor are additive. The tertiary structureof domain E of this triple mutant reveals how the interaction of all structural ele-ments involved in the spatial transformation of domain E of ERα influences theequilibrium of coexisting transcriptionally active and inactive forms. Althoughthe triple mutant shows significant transcriptional activity and domain E wascrystallized in complex with the agonist estradiol, the structure presents an

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alternative pattern for the stabilization of helix 12, which is surprisingly positionedin the coactivator binding groove as observed in the complexes of the domain Eof ERα with 4-hydroxytamoxifen and raloxifene. The structure reveals that themutations of C381S, C417S, and C530S influence the transcriptional activity ofERα due to different solvation properties of serine as compared to cysteine and thedisruption of hydrophobic interactions. All together, the three mutations loosen thestructure of those parts of domain E that are involved in the structural transitionof the transcriptionally active and inactive form.

Furthermore, structural principles of full antagonism versus partial agonismare elucidated in the crystallographic study of the heterodimer of domains E ofRARα in complex with the antagonist BMS614 and the mutant F318A of RXRα

in complex with oleic acid (7; Figure 5). The antagonist BMS614 carries a largeextension as compared to agonists of RARs. The crystal structure of the het-erodimeric complex reveals that this extension prevents the positioning of helix12 as observed in the transcriptionally active forms of domain E of NRs due tosteric interference. Helix 11 is tilted by about 11◦ toward the ligand-binding siteas compared to the position observed in domain E of RARγ in complex withall-transretinoic acid because the shape of BMS614 allows helix 11 to approachthe center of the ligand-binding site. Furthermore, helices 11 and 12 of RARα

are shortened, such that the accordingly lengthened loop 11–12 permits the posi-tioning of helix 12 in the binding groove of coactivators. Thus, the conformationof domain E of RARα in complex with BMS614 reveals the same mechanism ofantagonism as domain E of ERα in complex with either 4-hydroxytamoxifen orraloxifene.

The mutant F318A of RXRα revealed the presence of a copurified fortuitousligand in the crystal structure of the heterodimeric complex (7). The moleculewas identified as a long-chain fatty acid such as oleic, stearic, and palmitic acids.Oleic, stearic, and palmitic acids restore the transcriptional activation of reportergenes via RARαF318A inhibited by a weakly binding antagonist. Furthermore,oleic acid induces the binding of the coactivator TIF2 to either the mutant F318Aof RXRα alone or in complex with RARα bound to BMS614. Thus, oleic, stearic,and palmitic acids are characterized as weak agonists of RXRαF318A. However,domain E of RXRαF318A in complex with the copurified ligand presents helix 12located in the binding-site of coactivators as observed in domain E of NRs incomplex with antagonists such as raloxifene or BMS614. Helix 12 reaches thisbinding site owing to a shortening and particular positioning of helix 11 suchthat the axis of helix 11 is oriented roughly perpendicular to the axis of helix 10.Thus, helix 11 is localized similarly but not exactly as observed in the apo-formof domain E of RXRα. There is no explanation, which can be derived from thestructure, why RXRαF318A in complex with the copurified ligand does not as-sume the transcriptionally active form in the crystal. The discrepancy betweenthe biochemical characterization of long-chain fatty acids as weak agonists ofRXRαF318A and the localization of helix 12 in the binding-site of coactivatorsobserved crystallographically suggests that transcriptionally active and inactive

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forms of ligand associated RXRαF318A coexist in solution. Concentration andnature of transcriptional intermediary factors may influence the in vivo effects ofparticular ligands.

Experiments with PPARγ and GW0072 addressed the phenomenon of partialagonism in the case of ligands of PPARs (53; Figure 5). Both rosiglitazone andGW0072 induce the release of the corepressor NCoR, which is constitutively re-cruited by PPARγ . GW0072 competes with rosiglitazone for the ligand-bindingpocket; however, the relative efficacy of GW0072 to activate transcription of areporter gene is about 20% compared to the signal induced by rosiglitazone. Ac-cordingly, the recruitment of the coactivators CBP and SRC-1, by PPARγ , issignificantly reduced in response to GW0072 compared to the response elicited byrosiglitazone. The partial agonism of GW0072 observed in vitro and for the acti-vation of transcription in transient transfection assays does not suffice to convertpre-adipocyte and stem cell lines to mature adipocytes. GW0072 does not inducethe maturation of adipocytes, and it even efficiently inhibits the conversion to ma-ture adipocytes induced by rosiglitazone. The comparison of the crystal structuresof domain E of PPARγ , in complex with either GW0072 or rosiglitazone, revealsa different mode of binding for the ligands. GW0072 is positioned such that itdoes not interact directly with residues of helix 12 as observed for rosiglitazone.Consequently, the side chains of His-323, His-449, and Tyr-473, through whichthe imide group of rosiglitazone stabilizes the position of helix 12, adopt confor-mations close to the conformations observed in the apo-form of PPARγ . Thus,the partial agonism of GW0072 is based on a mode of binding that is sufficient todissociate corepressors from PPARγ , but does not permit the efficient stabilizationof domain E in the transcriptionally active form.

USP AND THE UNITING OF STRUCTURAL FEATURESOBSERVED IN TRANSCRIPTIONALLY ACTIVE ANDINACTIVE FORMS

In insects, ultraspiracle proteins (USPs) are the orthologs of RXRs of vertebrates.They are implicated in the reproduction, development, and metamorphosis ofinsects by activating the transcription of target genes upon heterodimerizationwith ecdysone receptors (EcRs), the transcriptional activity of which is controlledby binding of e.g. 20-hydroxyecdysone. Furthermore, USPs heterodimerize withDHR38, another NR of insects. Juvenile hormone was proposed as an endogenousagonist of USPs; however, this hypothesis is still debated such that no endogenousligand of USPs is unambiguously established yet (4 and references therein). Thealignment of the sequences of RXRs and USPs of the insect orders Lepidopteraand Diptera predicts that domain E of USPs assumes the antiparallelα-helicalsandwich fold without major insertions or deletions (4). The primary structuresdiverge chiefly in the loop connecting helix 5 and theβ-sheet and in the segment

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connecting helices 1 and 3. The sequence of the segment connecting helices 1and 3 is particularly well conserved in USPs of Lepidoptera. The tertiary structureof domain E of a lepidopteran USP (Heliotis virescensUSP; hvUSP) determinedby crystallography confirms the structural prediction of the sequence alignment(4). Moreover, the structure reveals the presence of a fortuitous copurified ligandthat was identified as a phospholipid composed of either phosphatidylglycerol orphophatidylethanolamine and two fatty acids of 18 and 16 carbon atoms, respec-tively. The copurified molecule is situated such that the hydrophobic chains of thefatty acids occupy the ligand-binding site in the region corresponding to the loca-tion of theβ-ionone ring of 9-cis retinoic acid in RXRα. The ligand-binding siteis not occupied by the copurified phospholipid toward the region corresponding tothe location of the carboxyl group of 9-cisretinoic acid in RXRα. The hydrophilichead group of the phospholipid protrudes out of the protein through a channel be-tween helix 11, helix 6, the segment between helices 1 and 3, and the N-terminusof helix 3. This channel probably represents the entrance to the ligand-binding siteof USPs.

The comparison of domains E of hvUSP, of the mutant RXRαF318A in theheterodimer with RARγ , and of RXRα either in complex with 9-cis retinoic acidor in the apo-form reveals that domain E of hvUSP unites structural features ob-served in the transcriptionally active and inactive forms of RXRα. Helices 4, 5,and 7 to 11 of hvUSP superimpose very well on the corresponding helices of do-main E of RXRα in complex with 9-cis retinoic acid. The positions of helices 1,3, 6 and theβ-sheet deviate significantly from the positions of the correspondingstructural elements of the transcriptionally active form of RXRα. The comparisonof domain E of hvUSP to the structure of domain E of apo-RXRα reveals thatthe position of the N-terminus of helix 3 is intermediary between its position inapo-RXRα and RXRα in complex with 9-cis retinoic acid. Thus, the hydrophilichead group of the phospholipid prevents the N-terminus of helix 3 from adoptingthe position as observed in the binary complex of RXRα and 9-cis retinoic acid.However, it does not force the N-terminus of helix 3 as far outwards as observedin apo-RXRα. The positioning of the N-terminus of helix 3 is accompanied by aparticular positioning of the segment connecting helices 1 and 3. The localizationof this segment is complemented by structural adaptations of the C-terminus ofhelix 3, loop 3–4, loop 8–9, and helix 1. Furthermore, the segment connectinghelices 1 and 3 adopts a conformation such that it interferes sterically with helix12, if helix 12 is modeled in the position as observed in structures representing theunique transcriptionally active form of NRs. In this manner, the segment betweenhelices 1 and 3 supports the positioning of helix 12 in the putative coactivatorbinding site as observed in the transcriptionally inactive form of RXRαF318A.The particular position of the segment connecting helices 1 and 3 is observedin this structure for the first time. The highly conserved sequence of this stretchmight indicate that its particular position as well as the deduced mechanism sup-porting the positioning of helix 12 in the coactivator binding site is unique toUSPs.

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In conclusion, domain E of hvUSP presents a transcriptionally inactive formthat unites structural features observed in both transcriptionally active and inac-tive forms of NRs. Such a compromise of conformations is observed for the firsttime; it might be induced by the copurified molecule and unique to lepidopteranUSPs. However, domain E of USPs underlines the polymorphic character of do-main E of NRs. Moreover, the binding mode of the copurified molecule suggeststhat endogenous ligands of USPs might enter the ligand-binding site through thechannel evinced between helix 11, helix 6, the segment between helices 1 and 3,and the N-terminus of helix 3. This supports further the mechanism of the structuraltransition of domain E of NRs upon binding of agonists.

HETERODIMERS OF RXRs

The structural analyses of domains E of the heterodimer between RARα and themutant RXRαF318A and of the heterodimer between RXRα and PPARγ presentprinciples of heterodimerization in comparison to homodimerization (7, 19). Ho-modimers of domain E of RXRs and PPARs are symmetric around a twofoldrotation axis parallel to the core of the dimerization interface formed by helix 10.Further, residues are located in helices 7, 9, and 11 as well as in loops 7–8 and9–10. These structural elements contribute to the dimerization in the same mannerfrom both molecules in homodimers. This symmetry is not maintained in the inter-face of the heterodimers of RXRs with either RARs or PPARs. In the heterodimerof RARα and RXRαF318A, helix 7 of RXRαF318A contributes an area that isfour times larger than the area contributed by helix 7 of RARα to the dimeriza-tion interface. The area provided by loop 8–9 of RARα is three times larger thanthe area provided by loop 8–9 of RXRαF318A. However, the same residues ofRXRα, which contribute to the formation of the RXR homodimer, are involved inthe stabilization of the heterodimer of RXRαF318A with RARα. The formation ofreciprocal but not symmetric salt bridges in the heterodimer of RXRα and PPARγdue to specific conserved sequences at the heterodimer interface constitutes a partof the structural basis for the asymmetry of heterodimers.

Two different types of RXR heterodimers are characterized. No response ofRXRs to agonists is detected in nonpermissive heterodimers formed with RARs,TRs, or VDR. Agonists will elicit a signal via RXRs in heterodimers formed withRARs if a ligand of RARs is present. However, RXRs can be activated by agonistsin permissive heterodimers formed with, for example, PPARs, LXR, and FXRirrespective of the presence of a ligand in the heterodimeric partner. Addition ofan agonist of PPARs results in an additive effect on transcriptional activation inthe case of RXR/PPAR heterodimers. The crystal structure of the heterodimer ofRXRα and PPARγ in complex with 9-cisretinoic acid, rosiglitazone, and peptidesderived from the coactivator SRC-1 suggests some features of the structural basisfor the permissiveness of RXR heterodimers. The interconnection of hydrogenbonds and salt bridges stabilizes the formation of a salt bridge between theε-amino

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group of Lys-431 in helix 10 of RXRα and the carboxylate group of Tyr-477, whichis the C-terminus of domain E of PPARγ . Thus, this salt bridge may permit thestabilization of helix 12 in the transcriptionally active form of PPARγ , even in theabsence of a PPAR agonist (19).

BINDING SITE OF COREPRESSORS ANDDISCRIMINATION OF TRANSCRIPTIONINTERMEDIARY FACTORS: RARs, TRs, and REV-ERBs

NCoR and SMRT are transcriptional intermediary factors by which RARs and TRactively repress transcription in the absence of agonists (23). Binding of agonistsdissociates the receptor/corepressor complexes and facilitates the formation ofreceptor/coactivator complexes. Antagonists of ERs and PR seem to require NCoRand SMRT in order to exert their anti-estrogenic activity. NCoR and SMRT sharea conserved bipartite NR interaction domain that contains two LXX I/H IXXX I/Lmotifs (28, 49, 54). The binding site of peptides containing this consensus motifwas mapped to overlap partially with the coactivator binding site of RARs andTR. The motif is predicted to be helical and to be at least one turn longer than theLXXLL motif of coactivators. Thus, the LXX I/H IXXX I/L motif is proposedto bind to RARs and TR such that its C-terminal part binds in the same locationas the LXXLL motif of coactivator fragments in the ternary complexes of PPAR,ER, and TR. The putative helix of the LXX I/H IXXX I/L motif is proposed tofit in this binding site such that its N-terminus prevents helix 12 from adoptingthe position observed in the transcriptionally active form of RARs and TR (54).Upon ligand-binding, the transcriptionally active form of domain E is stabilizedsuch that the position of helix 12 discriminates the LXX I/H IXXX I/L motif ofcorepressors and the LXXLL motif of coactivators owing to the different lengthsof theα-helical stretches, thus providing the structural basis for the dissociationof corepressors and the recruitment of coactivators (49, 54).

The homologs Rev-erbA and RVR (Rev-erbB) are orphan NRs that constitu-tively repress transcription employing NCoR as a cofactor (13). Their sequencescontain a significant insertion between helix 1 and helix 3 of domain E, whichprobably folds into a small supplementary domain. The sequences of Rev-erbs donot contain helix 12 with the conserved core of the transcriptional AF-2, whichexplains their impotence to activate transcription. Homology modeling shows thatthe putative ligand-binding site is partly collapsed owing to the absence of helix 12.Furthermore, the remaining putative ligand-binding site is filled up by large hy-drophobic side chains (59). This suggests that Rev-erbs may not respond to endoge-nous ligands. Because the role of agonists is to assemble the complete functionalAF-2 by bringing together helix 12 (the dynamic part of AF-2) and the cornerbetween helices 3 and 4 (the static part of AF-2), the loss of a complete and func-tional transcriptional AF-2 renders a ligand useless. The corepressor interaction

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Figure 10 Proposed differential binding of corepressors and coactivators to NR LBDs.A) In the absence of ligand, the corepressor is bound to the LBD (H3, H4, and H11),the “activation helix” H12 being away.B) Upon ligand binding, H12 is repositioned to alocation that is no longer compatible with corepressor binding, which is therefore released.The newly formed AF-2 surface (H3, H4, and H12) can now recruit coactivators (adaptedfrom 59).

regions, which were mapped to the corner between helices 3 and 4 and to helix 11,form a large continuous hydrophobic surface in the homology model of both ho-mologs. Point mutations of residues contributing to the hydrophobic surface affectthe binding of corepressors and the silencing of transcription via both homologs.This corepressor binding site overlaps with the binding site of coactivators as itwas identified previously in other NRs, thus suggesting that the general role ofagonists is to remodel this surface, and thus, to promote corepressor release andcoactivator recruitment (59; Figure 10). Thus, Rev-erbs share the structural basisof corepressor recruitment with other NRs. However, the principles underlying theregulation of their repressive activity are not yet entirely established.

CONCLUSION

Domain E harboring the ligand-binding site and ligand-dependent transcription ac-tivation function AF-2 is indispensable for endocrine, paracrine, and intracrine reg-ulation exerted by NRs in homeostasis and development. The increasing number of

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complexes with different types of ligands as well as new structures of further repre-sentatives revealed details of ligand-receptor recognition and allowed refinement ofthe functional mechanism of NRs. Homology modeling, assisted by site-directedmutagenesis, can present fairly accurate pictures of ligand-receptor complexes;however, the explanation of phenomena such as the superagonistic activity of20-epi ligands of VDR requires experimental structural approaches. Domain E ofVDR in complex with either its natural ligand or 20-epi superagonists stronglysuggests that the transcriptionally active form of all NRs is principally the same.However, the ensemble of structures of NRs, in particular of ERs and PPARs,reveals structural polymorphism of NRs and underlines the close proximity ofthe mechanisms of receptor activation upon ligand-binding and principles of pro-tein folding. Thus, the latest results in the structural biology of NRs evince thatligands tip the scales of the equilibrium of conformational populations such thatfull agonists stabilize a unique transcriptionally active form of the receptor andthat antagonists prevent the adoption of this transcriptionally active form. Par-tial agonism at the structural level is based on the insufficient stabilization of thetranscriptionally active form.

Investigations closely related to the structural basis of the transformation ofdomain E upon ligand-binding concern the regulation of transcription by orphanreceptors. Considering the complexity of the regulation of transcription by NRs, itcannot be excluded that some orphan receptors do not require ligands in order toparticipate in the control of gene expression as suggested for Rev-erbs. Structuralbiology will contribute to reveal the mechanisms by which orphan receptors joinin the regulation of transcription.

Thus, the entire functional mechanism of NRs has further dimensions than onlythe binding of ligand. The crystal structure of an entire NR is desirable, and thesearch for crystallization conditions stabilizing larger entities of NRs sufficiently toinduce crystallization will continue. Data suggest that antagonists of ERs implicatedomain F to exert repression of transcription via interaction with corepressors(46a). Consequently, the structural basis of the interaction of domains E and Fin the presence and absence of ligands raises new interest. If domain F of ERsindeed plays an endogenous role in the repression of target genes (dependingon the binding of antagonists), the question arises: Which molecules are naturalantagonists of ERs? Until recently, no natural antagonists of NRs were established.The constitutive androstane receptor (CAR) is the first NR for which it was shownthat transcriptional activity is attenuated by the binding of steroid metabolites,indicating that an entire new facet of the hormonal system may be revealed in thefuture (18).

The structures of ternary complexes, including small fragments of coactiva-tors, provide an initial understanding of the structural determinants underlying theexchange of corepressors and coactivators. However, the determination of com-plexes, including entire coactivator or corepressor molecules, are needed at lowand high resolution in order to refine our understanding of the spatial and temporalassembly of transcription intermediary factors and the significance of structural

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polymorphism of NRs. Furthermore, the three-dimensional interplay of domainsA/B and E in the regulation of transcription, which is suggested by the synergismof AF-1 and AF-2 in ERα, needs to be addressed (10, 21, 37, 64). Finally, structuresof complexes providing the link to the basal transcriptional machinery are neededin order to complete our understanding of the structural basis of the regulation oftranscription.

ACKNOWLEDGMENTS

We cordially thank Monique Gangloff, Natacha Rochel, Marc Ruff, and GiuseppeTocchini-Valentini for entrusting their manuscripts to us before publication. Weare grateful to Pascal Egea for providing us with Figure 7.

Visit the Annual Reviews home page at www.AnnualReviews.org

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April 24, 2001 14:50 Annual Reviews AR128-14-COLOR

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Figure 4 (from previous page) Diagrams of domain E of representative NRs. Protein= green; helix 3= blue; helix 4= pink; ligands= yellow; helix 12 = red; LXXLLmotif of co-activators= violet. Helices are numbered 1 to 12 as reported for the first NRstructure, RXRα. Throughout the text, we refer for all NRs discussed to the numbering ofhelices 1 to 12 as labelled inA. (A) The apo-form of RXRα, the binding-site of which is notaccessible to ligand (PDB entry code 1lbd). (B) The binary complex of RARγ and all-transretinoic acid in the transcriptionally active form (PDB entry code 2lbd). (C) The ternarycomplex of ERα, distilbestrol, and a fragment of the coactivator GRIP, which contains theLXXLL motif (PDB entry code 3erd). This structure represents the transcriptionally activeform of NRs and indicates the binding-site of coactivators. (D) The binary complex of ERαand the selective ER modulator tamoxifen (PDB entry code 3ert). This structure representsa transcriptionally inactive form of NRs where helix 12 is located in the binding-site ofcoactivators. (E) The binary complex of ERβ and the partial agonist genistein (PDB entrycode 1qkm). In the crystal structure, helix 12 is located in the coactivator binding groove.Helix 12 as observed in the transcriptionally active form of NRs is superimposed in order toillustrate the alternative positioning of helix 12 in complexes of NRs with partial agonistsas underlined by the red double pointed arrow.

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Figure 7 The effect of the binding of 9-cisretinoic acid on the tertiary structure of domainE of RXRs. The models of domain E of apo-RXRα and the binary complex of RXRα and 9-cisretinoic acid were superimposed (PDB entry codes 1lbd and 1fby, respectively). DomainE of apo-RXRα is depicted in green and yellow, and domain E of the binary complex inblue and red. The gray arrows underline the structural rearrangement of helices 11, 12, andthe N-terminus of helix 3 (adapted from 15).

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Figure 9 (from previous page) Simulations of the escape of all-trans retinoic acid fromdomain E of RARγ (adapted from 5). (A) Enhanced molecular dynamics simulation ofligand escape. The initial phase of 20 copies of all-trans retinoic acid leaving domain Eof RARγ is depicted at three steps along the trajectory: t= 0, t = 37.5 ps, t= 73.5ps (from the left to the right). The protein is depicted as a ribbon and cylinder diagram.The core of the protein is depicted in green, helix 3 in gray, helix 4 in orange, helix 12 inyellow, theÄ-loop in blue, and theβ-sheet in red. Helices 10 and 11 are depicted as yellowcylinders. All-transretinoic acid is drawn as a white and blue ball-and-stick representation.The simulation suggests that helix 12 opens the entrance of the ligand-binding site in orderto allow the ligand to leave domain E. (B) A path for the structural transition of domain E ofRARγ during the escape of the ligand calculated by the conjugate peak refinement method.The protein is represented as a series of fourα-carbon traces; all-trans retinoic acid isrepresented as a stick diagram in the corresponding colors. Snapshots are taken in the orderof models: blue, green, yellow, and red. The blue model represents the transcriptionallyactive form observed in the crystal structure. The protein models are truncated in order toreveal the straightening of helix 3 and the concomitant rearrangement of theÄ-loop (left)as well as the rearrangement of helix 11 and loop 11-12 (right).

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Annual Review of Biophysics and Biomolecular Structure Volume 30, 2001

CONTENTSHYDROGEN BONDING, BASE STACKING, AND STERIC EFFECTS IN DNA REPLICATION, Eric T. Kool 1STRUCTURES AND PROTON-PUMPING STRATEGIES OF MITOCHONDRIAL RESPIRATORY ENZYMES, Brian E. Schultz, Sunney I. Chan 23

MASS SPECTROMETRY AS A TOOL FOR PROTEIN CRYSTALLOGRAPHY, Steven L. Cohen, Brian T. Chait 67A STRUCTURAL VIEW OF Cre-loxP SITE-SPECIFIC RECOMBINATION, Gregory D. Van Duyne 87PROBING THE RELATION BETWEEN FORCE--LIFETIME--AND CHEMISTRY IN SINGLE MOLECULAR BONDS, Evan Evans 105NMR PROBES OF MOLECULAR DYNAMICS: Overview and Comparison with Other Techniques, Arthur G. Palmer III 129STRUCTURE OF PROTEINS INVOLVED IN SYNAPTIC VESICLE FUSION IN NEURONS, Axel T. Brunger 157AB INITIO PROTEIN STRUCTURE PREDICTION: Progress and Prospects, Richard Bonneau, David Baker 173

STRUCTURAL RELATIONSHIPS AMONG REGULATED AND UNREGULATED PHOSPHORYLASES, Jenny L. Buchbinder, Virginia L. Rath, Robert J. Fletterick 191

BIOMOLECULAR SIMULATIONS: Recent Developments in Force Fields, Simulations of Enzyme Catalysis, Protein-Ligand, Protein-Protein, and Protein-Nucleic Acid Noncovalent Interactions, Wei Wang, Oreola Donini, Carolina M. Reyes, Peter A. Kollman 211CHAPERONIN-MEDIATED PROTEIN FOLDING, D. Thirumalai, George H. Lorimer 245INTERPRETING THE EFFECTS OF SMALL UNCHARGED SOLUTES ON PROTEIN-FOLDING EQUILIBRIA, Paula R. Davis-Searles, Aleister J. Saunders, Dorothy A. Erie, Donald J. Winzor, Gary J. Pielak 271

PHOTOSYSTEM II: The Solid Structural Era, Kyong-Hi Rhee 307BINDING OF LIGANDS AND ACTIVATION OF TRANSCRIPTION BY NUCLEAR RECEPTORS, Anke C. U. Steinmetz, Jean-Paul Renaud, Dino Moras 329PROTEIN FOLDING THEORY: From Lattice to All-Atom Models, Leonid Mirny, Eugene Shakhnovich 361STRUCTURAL INSIGHTS INTO MICROTUBULE FUNCTION, Eva Nogales 397PROPERTIES AND BIOLOGICAL ACTIVITIES OF THIOREDOXINS, Garth Powis, William R Montfort 421

RIBOZYME STRUCTURES AND MECHANISMS, Elizabeth A. Doherty, Jennifer A. Doudna 457

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Binding of Ligands and Activation of Transcription by Nuclear Receptors

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