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Mechanism of the nuclear receptor molecular switch
Review
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Vol.29 No.6 June 2004
Mechanism of the nuclear receptor molecular switch Laszlo Nagy1 and John W.R. Schwabe2 1
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Nagyerdei Ko¨ru´t 98, Debrecen H-4012, Hungary 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Nuclear receptors are central to the regulation of development, endocrine signalling and metabolism. The transcriptional activity of many receptors is controlled through the binding of small, fat-soluble molecules to the ligand-binding domain. In most cases, ligand binding turns the receptors into potent activators of transcription. This switch involves the exchange of co-regulator proteins that mediate transcriptional regulation. Structural and biochemical studies have together revealed the mechanism of action of this ligand-induced molecular switch, in which changes in the dynamic behaviour of the receptor play a key role. This remarkable dynamic mechanism has facilitated the evolution of a family of nuclear receptors with highly diverse ligand recognition and signalling properties. The regulation of gene expression requires the ability to switch between two opposing, mutually exclusive or redundant processes. This is essential for the regulation of growth, development and homeostasis in complex multicellular organisms, which use hormones and metabolites to act as messengers that signal the status of the organism as a whole. Fat-soluble hormones and intermediary metabolites are particularly well suited to mediate intercellular communication because, unlike water-soluble peptide hormones, they can pass freely through the lipid bilayer of the cell membrane [1]. Such molecules have been exploited by nature to function as regulatory ligands for a major class of transcriptional regulators called nuclear receptors. These nuclear receptors are special in that they deliver hormonal or metabolic signals directly to the genome by activating or repressing gene expression. In principle, this regulatory system does not require – although it can interact with – additional second messenger systems or protein cascades to exert its effects on transcription. Nuclear receptors were first identified nearly 40 years ago as intracellular receptors for some steroids [2,3]. More than 20 years passed, however, before it became apparent that these steroid receptors are part of a superfamily of metazoan transcription factors that evolved before the divergence of vertebrates and invertebrates and that share a common domain structure consisting of separate DNAand ligand-binding domains [4– 6] (Box 1). The 48 human Corresponding authors: Laszlo Nagy ([email protected]), John W.R. Schwabe ([email protected]). Available online 6 May 2004
members of this family include both receptors for which ligands are known and ‘orphan receptors’ for which there are, as yet, no known ligands [7]. As our understanding of nuclear receptors has grown, it has become increasingly evident that nuclear receptors form a very diverse family in terms of both physiological role and molecular action. The classical view derived from endocrine receptors is that they are each activated by a unique high-affinity ligand with a dissociation constant in the nanomolar range. The best examples of these receptors are members of the steroid hormone receptor family such as the oestrogen receptor (ER) and the androgen receptor. More recently, so-called ‘metabolic receptors’ have been identified that are activated by abundant but low-affinity ligands with dissociation constants in the micromolar range. For example, peroxisome proliferator-activated receptors (PPARs) are activated by various fatty acids, eicosanoids and prostanoids, and liver X receptors (LXRs) are activated by oxysterols [8]. It has also emerged that Box 1. Nuclear receptor domains Nearly all nuclear receptors have two conserved domains (Figure I): a DNA-binding domain (DBD) and C-terminal ligand-binding domain (LBD). A few receptors lacking a DBD have been identified, however, such as the orphan nuclear receptor short heterodimer partner (SHP). Owing to the constraints of maintaining structure and function in a small domain, the DBD is very highly conserved. At its carboxyl terminus is a nuclear localization signal required for nuclear entry; it has been also proposed that a nuclear export signal is located within the DBD itself [64]. The DBD contains two zinc-binding motifs that maintain the domain architecture (reviewed in Refs [65,66]) and have evolved together as a single functional unit. As a result of extensive structural and biochemical investigations, the mechanisms of targetsite recognition by nuclear receptor DBDs are well understood (reviewed in Ref. [67]). Nuclear receptor LBDs are more diverse and are discussed more extensively in the main text. The LBD is primarily helical and has a partly rigid, partly mobile character that might have facilitated rapid evolution [68].
AF-1 <----------------> N–
NES NLS ^ ^ DBD
AF-2 <------------------------> –C LBD Ti BS
Figure I. Common domain structure of nuclear hormone receptors. The N and C termini are indicated. Abbreviations: AF-1, activation function 1 (ligand independent); AF-2, activation function 2 (ligand dependent); DBD, DNA-binding domain; LBD, ligand-binding domain; NES, nuclear export signal; NLS, nuclear localization signal.
www.sciencedirect.com 0968-0004/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.04.006
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nuclear receptors can both activate and repress transcription. The role of the ligands can be to activate inactive or repressed receptors, to deactivate or superactivate constitutively active receptors, or, in a few cases, to serve even as non-regulatory structural co-regulators. Finally, it has emerged recently that some receptors apparently lack a ligand binding cavity and therefore might be regulated by other means. The diverse developmental and physiological roles of nuclear receptors are perhaps best illustrated by two examples: the retinoid X receptor (RXR) and the retinoic acid receptor (RAR), which are activated by metabolites of vitamin A and play a central role in the regulation of developmental pathways by through the induction of crucial embryonic Hox genes [9,10]. In a different biological setting, the receptors for some fatty acids (PPARs), bile acids [farnesoid X receptor (FXR); also known as bile acid receptor (BAR)] or cholesterol metabolites (LXRs) regulate complete metabolic pathways such as the uptake, oxidation, storage and intracellular processing of extracellular lipids (reviewed in Ref. [8]). In this way, the metabolic state of the organism programmes the metabolic pathways of a cell through specific nuclear receptors. Because of their diverse and important biological roles, nuclear receptors are primary targets of drug discovery. Furthermore, several receptors are associated with human diseases: for example, RARs are associated with types of leukaemia [11], the ER with breast cancer growth [12], glucocorticoid receptors with inflammation control [13], and PPARs with metabolic diseases such as diabetes [14]. It is evident that elucidating the mechanisms of nuclear receptor activation has widespread implications for our understanding of biological regulation, as well as for efforts to exploit nuclear receptors as drug targets. It is therefore highly significant that the mechanism through which nuclear receptor ligands facilitate the switch from an inactive or repressive condition to an active state has recently become clear through a combination of structural and biochemical techniques. In this review, we describe the studies that have led to our present understanding of the mechanism of the nuclear receptor switch. Specific recognition of a small-molecule ligand There are now numerous structures of nuclear receptor ligand-binding domains (LBDs) bound to small molecules [15]. Such molecules include the natural ligands of the receptor and synthetic agonists and antagonists, as well as adventitiously bound molecules that resemble the natural ligands. By contrast, there are rather few structures of an LBD in which there is no small molecule bound. The first thing to note from the structures is that all of the LBDs share a common, primarily helical, structural scaffold that forms a single protein domain [16]. This domain can be described as a three-layer a-helical sandwich and can be divided into two halves (Figure 1a). In the lower half of the domain there is no central helical layer. Instead, there is a large non-polar cavity in which the various ligands bind. The back and the front of this cavity are sealed by, respectively, a two-stranded b-sheet and the carboxy (C)-terminal helix of the receptor (termed www.sciencedirect.com
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helix 12 on the basis of the structure of apo-RXR [17]). Within this common basic framework, there are several areas where the structure can differ according to the receptor, in particular, the region between helices 1 and 3. Nuclear receptor ligands share in common a mainly non-polar character; however, they vary greatly in size and detailed stereochemistry (Box 2). This variation constitutes the recognition challenge faced by the various LBDs. An important issue, therefore, is how this conserved structural domain can adapt to fit such a wide range of ligands. As one might expect, the diversity in ligand size and shape is matched by a similar diversity in the LBDs themselves. The interior surface of the ligand-binding pockets is mostly made up from non-polar amino acids and thereby matches the character of the various ligands. Specificity is achieved through a limited number of stereo-specific polar contacts and the actual shape of the ligand-binding cavity. It is notable that the receptors that bind with high affinity to a specific ligand, such as the thyroid hormone receptor (TR) and ER, have small ligandbinding cavities [18,19]. Receptors that are apparently more promiscuous and can bind numerous ligands but with lower affinity, such as PPARg and the pregnane X receptor (PXR), have much larger ligand-binding cavities that can accommodate the various ligands in different orientations [20– 23]. It is remarkable that this common structural framework can generate such a vast array of shapes and chemistries of the ligand-binding pocket. The volume of ˚3 this pocket ranges from zero to more than 1000 A (Figure 1b). The larger cavities, such as those in PXR, Ultraspiracle and PPARg, are created by an opening up of the helical framework [20 – 22,24,25]. By contrast, the would-be cavities in the orphan nuclear receptors Nurr1 [26] and DHR38 [27] are completely filled by bulky hydrophobic side chains and a tightening of the helical framework. This lack of a cavity suggests that these receptors are not regulated by conventional ligand binding and either have constitutive activity or are regulated by another mechanism. As mentioned above, only a few structures of a nuclear receptor LBD without a small-molecule ligand have been determined. The first of these was the structure of apo-RXR [17]. Comparison of this structure with the ligand-bound RAR suggested that receptors undergo a very specific switch between two conformations, which involves a major rearrangement of helices 10/11 and 12, as well as more subtle changes [28]. It has subsequently emerged, however, that apo-RXR might be unusual (perhaps because it forms a tetramer in the absence of ligand [29]), because the structures of several other receptors without bound ligand, including PPARg, PXR and liver receptor homologue-1 (LRH-1), closely resemble the ligand-bound state [20,22,30]. These structures suggest that the mechanism of the molecular switch might be more subtle in the latter receptors than it is in RXR. Co-regulators for recruiting large complexes A chief goal of the nuclear receptor field has been to identify the mechanisms and proteins involved in mediating the activation or repression of the receptor itself: in other
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H1
90o
H10/11 H12 H3 (b)
Nurr1 (0 Å3)
TRβ (~600 Å3)
PXR (~1100 Å3) Ti BS
Figure 1. Structural framework of nuclear receptor ligand-binding domains (LBDs). (a) The LBD is formed from a three-layer a-helical sandwich. The layers are shown in blue, yellow and red. In the lower portion of the structure the central helical layer is absent, creating a mainly non-polar cavity in which the ligand (green) binds. Helix 12 and a b-strand (magenta) close the front and back of the ligand-binding cavity, as can be seen on the left. The structure shown is that of the retinoic acid receptor-g (RARg) bound to all-trans retinoic acid (PDB code 2LBD) [28]. (b) Structures of the LBD of Nurr1, the thyroid hormone receptor-b (TRb) and the pregnane X receptor (PXR; PDB codes 1OVL, 1BSX and 1ILG, respectively) [18,22,26]. The ligand-binding cavity is illustrated by a blue mesh: Nurr1 has no cavity; the cavities in TRb and PXR are about 600 and 1100 A˚3, respectively. Note that the size and position of the cavity is dependent upon the extent of opening up of the helices in the lower portion of the structure.
words, how does ligand binding translate into regulation of transcription? This question remains to be answered in full but, at least from the point of view of the receptors, we now have a good understanding of the next step in the signalling process. This progress has been the result of the identification of proteins that interact specifically with either ligand-bound or ligand-free nuclear receptors (reviewed in Refs [31,32]). These proteins are called either co-activator or corepressor proteins, depending on the transcriptional outcome. In general, co-repressors bind to ligand-free nuclear receptors [33 – 35] and are displaced by activating ligands. (Note that some antagonists also displace co-repressors, others are compatible with co-repressor binding, and the so-called ‘inverse agonists’ actually enhance co-repressor binding.) After ligand-binding and displacement of corepressor, co-activator proteins associate with the receptor LBD [36 –39]. After co-regulator proteins were identified, the race was on to establish which part of the co-regulator interacts with the receptor and to identify the interaction surface on the receptor. What has emerged from these studies is a comparatively straightforward mechanism of co-regulator selection. Many co-activators and co-repressors contain multiple, short, receptor interaction motifs, the sequences of which can be generalized as LxxLL in co-activators www.sciencedirect.com
[40,41] and LxxxIxxx[I/L] in co-repressors [42 – 45]. Mutational studies to map the region on the surface of the LBD that interacts with co-activators and co-repressors yielded what seemed at first to be a rather surprising result: that is, co-repressors and co-activators interact with largely overlapping surfaces on the LBD [42 – 47]. Of course, such overlap provides the advantage that binding of the two factors is mutually exclusive and that the only thing that the receptor has to do is to choose which factor can bind. This choice of co-regulator seems to be the switch – in its simplest interpretation – that is controlled by ligand binding. Structural studies of receptor complexes with coactivator peptides have shown that the LxxLL co-activator motif adopts a helical structure on binding to a hydrophobic groove on the surface of the LBD [20,41,48] (Figure 2). The peptide makes contacts with helices 3, 4 and 12. There is a particularly important charge clamp, formed by a lysine and glutamate in helices 3 and 12, respectively, that interacts with the helix dipole. From these structures, it is evident that a particular position of helix 12 is essential to support co-activator binding. It is therefore especially striking that structures of antagonistbound ER show that the inhibitory ligand not only causes helix 12 to be displaced from the position required for co-activator binding, but also itself occupies the
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Box 2. The diversity of nuclear receptor ligands Nuclear receptors can be activated by various fat-soluble molecules, including ‘classical’ steroid hormones such as oestradiol for the oestrogen receptor (ER), cortisol for the glucocorticoid receptor, progestin for the progesterone receptor and testosterone for the androgen receptor. All of these ligands are the products of wellcharacterized steroid hormone biosynthetic pathways. Some vitamins also exert their effects through nuclear receptors. Vitamin-A-derived all-trans retinoic acid and 9-cis retinoic acid function through the retinoic acid (RAR) and retinoid X (RXR) receptors, whereas vitamin D3 activates the vitamin D receptor (VDR). Among intermediary metabolites, an increasing number of mainly low-affinity ligands have been found for previously identified orphan receptors. These metabolites include fatty acids (polyunsaturated fatty acids, oxidized fatty acids), arachidonates and prostanoids for
(a) Chenodeoxycholate
peroxisome proliferator-activated receptors (PPARs), oxysterols for liver X receptors (LXRs) and bile acids for the farnesoid X receptor [FXR; also known as bile acid receptor (BAR)]8. Some receptors, such as pregnane X receptor (PXR), steroid and xenobiotic receptor (SXR), and constitutive androstane receptor (CAR), are even activated by xenobiotics [69]. How xenobiotics can exert evolutionary pressure on these receptors is particularly intriguing. The ability of nuclear receptors to bind to different, structurally distinct ligands (Figure I) is reflected in the structure of their ligandbinding domain (LBD). Unlike classical hormone receptors with tight-fitting LBDs (such as ER and RAR), PPARs and LXRs have large cavities that are only partially filled by ligand [20,70], which explains how more than one type of molecule can regulate their activity.
(c) All-trans retinoic acid
(b) 17β-oestradiol
(d) Rifampicin CH3
CH3
OH COOH
OH
CH3
O
O OH CH3
COOH
O
CH3
OH
CH3 NH
CH3
N
H3CO N OH
OH
N
O
OH
OH
O O CH3
Ti BS
Figure I. Chemical structures of nuclear receptor ligands. (a) The bile acid receptor ligand, chenodeoxycholate (a primary bile acid and a metabolite of cholesterol metabolism). (b) The oestrogen receptor ligand, 17b-oestradiol (a female sex hormone). (c) The retinoic acid receptor ligand, all-trans retinoic acid (a derivative of vitamin A and a potent morphogen). (d) The xenobiotic pregnane X receptor ligand, rifampicin (an antibiotic used in the treatment of tuberculosis).
co-activator-binding groove between helices 3 and 4 [19,48] (Figure 2). This has led to the suggestion that co-repressor peptides might bind to the surface of the LBD in a fashion very similar to that of the coactivators, but that the longer motif might form an extra helical turn such that it does not require helix 12 to be in the active position [42 – 44]. So far, unfortunately, there have been no structures of complexes between ligand-free receptors and co-repressor peptides; however, the structure of antagonist-bound PPARa with a co-repressor peptide has been determined [49]. This structure shows, as predicted from the mapping studies, that the co-repressor peptide does indeed bind in the same hydrophobic groove as the co-activator, with helix 12 displaced from the active position. LBDs as molecular sensors The structural studies discussed above have revealed much of the basis of specific ligand recognition and the nature of the various co-regulator complexes. There is a difference, however, between understanding the various structures and understanding how the molecular switch functions – in other words, the mechanism through which the ligands cause a switch in the choice of co-regulator. The problem is that the structures do not provide a clear explanation for why, in the ligand-free receptor, helix 12 cannot adopt the active position or lead to the recruitment of co-activator proteins. Indeed, in several of the ligandfree structures helix 12 does in fact occupy the active www.sciencedirect.com
position, raising the question of how co-repressor proteins are able to bind to apo-receptors [20,22]. Interestingly, in the complex of PPARa and co-repressor, a synthetic antagonist prevents helix 12 from adopting the active conformation, and thus facilitates co-repressor binding and can be considered an inverse agonist [49]. In summary, it is clear that by themselves the structural studies do not fully clarify the mechanism of the nuclear receptor molecular switch. Fortunately, however, crystallographic studies of nuclear receptors have been complemented by other biochemical and biophysical studies, which have proved to be more fruitful in terms of clarifying the differences between ligand-bound and ligand-free receptors. Proteolytic sensitivity assays show that apo-LBDs are generally more sensitive to proteolysis [50,51]. Gel-filtration analyses suggest that ligand binding slightly reduces the apparent size of the LBD. Thermal denaturation assays, monitored by either circular dichroism or differential scanning calorimetry, show a marked increase in the melting temperature of the LBD on ligand binding [23]. Finally, dynamic stabilization assays investigating the interaction between the isolated helix 1 and the remainder of the LBD show that there is a significant stabilization of this interaction on the addition of ligand (and also co-regulators) [52]. Taken together, these results strongly suggest that ligand binding stabilizes the receptor LBD, resulting in a more compact and rigid structure.
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(a)
ERα + agonist
ERα + agonist + CoA
ERα + antagonist
ERβ + antagonist
(b)
Apo-PPARγ 1
Apo-PPARγ 2
PPARα + agonist + CoA
PPARα + antagonist+ CoR
Figure 2. Effect of ligands and co-regulators on the position of helix 12. The top row of structures shows the oestrogen receptor (ER) bound to agonists and antagonists (PDB codes 1ERE, 1ERR, 1GWR and 1QKM) [19,63]. Note that in the antagonist-bound structures helix 12 adopts a position very similar to that of the co-activator in the agonist-bound structure. The bottom row of structures shows peroxisome proliferator-activated receptors (PPARs) in the absence of ligand (left two structures; PDB code 1PRG), bound to agonist and co-activator (PDB code 2PRG) and to antagonist and co-repressor (PDB code 1KKQ) [20,49]. Note that in the absence of ligand, helix 12 can adopt both the active position and an alternative position. In the presence of antagonist and co-repressor, helix 12 unravels. ERs and PPARs are shown in light blue and green, respectively; helix 12 in yellow; agonists and antagonists in magenta and cyan, respectively; and co-activator and co-repressor peptides in red and dark blue, respectively.
This conclusion is complemented by two further experimental observations. First, the crystal structure of apo-PPAR-g shows a striking distribution of crystallographic temperature factors [20] (Figure 3a), which seems to suggest that the lower portion of the LBD, as seen in Figure 3a, is less rigid than the upper portion. This idea is further supported by NMR studies of apo- and holoPPARg and PPARa, which found that essentially the same region of the ligand-free LBD shows exchange broadening on a millisecond timescale [53,54], suggesting that there is significant conformational mobility but on a timescale slower than the overall tumbling of the protein. Altogether, these findings fit with suggestions that the lower portion of the receptor is rather dynamic and might have some of the properties of a molten globule [55]. The dynamic properties of the apo- and holo-receptors are thus distinct. In itself, however, this difference does not explain how the choice of co-regulator binding is controlled. The key to the binding choice seems to be the dynamic behaviour of helix 12 of the receptor. Fluorescence anisotropy techniques show that helix 12 is very dynamic in apo-PPARg, with motions on the scale of a few nanoseconds, which suggests that it has mobility independent of the rest of the LBD [56]. On ligand binding, helix 12 shows significantly slower dynamics, suggesting that it is bound to the surface of the LBD, presumably in the active conformation [56]. Similar studies of the ER www.sciencedirect.com
containing a fluorescent label near the C-terminus of helix 11 support the finding that ligand binding has a profound effect on the fast motions of this region of the LBD [57]. Ligand binding probably stabilizes helix 12 in the active conformation in two ways. First, in PPARg, as in many other receptors, the ligand itself makes direct contact with residues in helix 12, thereby promoting the active conformation. Second, the ligand globally stabilizes the lower half of the LBD, and this in itself will favour helix 12 stably adopting the active conformation. Of course, both of these mechanisms also promote the binding of co-activator peptides. In the absence of ligand, the larger co-repressor interaction motif can bind to the LBD and partially stabilize the ligand-free conformation, as observed in the dynamic assembly assay [52]. It is also important to note that the relative amount of co-repressor and co-activator has been shown to influence the equilibrium between the active and inactive conformations [58]. In summary, it is clear that the ligand-free LBD of PPARg is partly rigid and partly mobile. Helix 12 of the receptor is especially mobile, showing fast, independent, segmental motion [56]. Ligand binding globally stabilizes the LBD and, through both this and direct contacts, stabilizes helix 12 in the active conformation, which in turn promotes co-activator binding. This global stabilization can be perhaps considered as the switch mechanism,
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(a) Upper portion Tightly packed, three-layer helical sandwich Lower crystallographic temperature factors Sharp peaks in NMR spectra Forms rigid support for the apo-LBD Helix 12 Short C-terminal helix of LBD Fast segmental mobility (nanosecond timescale) Dynamic behaviour sensitive to different classes of ligand Lower portion Helices framing ligand-binding cavity Higher crystallographic temperature factors Conformationally labile (millisecond timescale) Dynamic character analogous to molten globule (b) Basal transcriptional activity Helix 12 mobility and LBD dynamics
RARα
PPARγ
LRH-1 Ti BS
Figure 3. Nuclear receptor dynamics and activity. (a) Structure of a ligand-free peroxisome proliferator-activated receptor (PPARg; PDB code 1PRG) [20] coloured according to crystallographic temperature factors and annotated according to our understanding of the dynamic properties of this molecule [53,56]. (b) Different nuclear receptors are likely to show different degrees of dynamic lability that correlate with their basal transcriptional activity. For example, liver receptor homologue-1 (LRH-1) has constitutive activity and is stable enough to be crystallized in the absence of ligand; retinoic acid receptor-a (RARa) is a repressor in the absence of ligand and crystallization attempts without ligand have been unsuccessful, which is consistent with the idea that the ligand-binding domain (LBD) is conformationally mobile; peroxisome proliferatoractivated receptor-g (PPARg) adopts an intermediate position between these extremes.
with helix 12 acting as the sensor through which the state of the receptor is read by co-regulators. The next question is whether this mechanism, established for PPARg, applies to other nuclear receptors, especially because nuclear receptors are rather diverse in their mechanism of action. The available evidence suggests that nuclear receptors show a great range in dynamic behaviour that correlates rather well with their level of basal activity. It seems likely, therefore, that there is a stability scale on which the nuclear receptors adopt different positions (Figure 3b). The LBDs of LRH-1 and Nurr1 are constitutively active. Both of these have been crystallized in the absence of ligand, revealing stable structures with helix 12 in the active conformation [26,30]. By contrast, ligand-free TR and RAR are strong repressors, have proved so far to be refractory to crystallization efforts, and show rather low melting temperatures in the absence of ligand. Thus, TR and RAR probably sit at the more dynamic end of the scale. PPARg sits between these two extremes, with a rather high basal activity but with the ability to be activated by ligand. Concluding remarks The realization that the dynamic behaviour of the LBD is key to the mechanism of the molecular switch explains some hitherto puzzling observations about nuclear receptors. In particular, it explains how selective modulators can fine-tune the activity of the receptor [57]. Hence, the switch is not like a regular on – off light switch, but is more like a dimmer switch. This in turn explains why some receptors, such as RAR-related orphan receptor-b (RORb) and constitutive androstane receptor (CAR), seem to www.sciencedirect.com
function in the reverse direction, with ligands serving as deactivators [59,60]. But several key questions remain unanswered. It is still not fully clear how co-repressors bind to apo-receptors. The only available structure – that of PPARa bound to a SMRT (silencing mediator of RAR and TR) peptide – is in the presence of an antagonist ligand, which is likely to stabilize the LBD structure. The structure of a ligandfree repressing receptor, such as RAR, TR or LXR, bound to co-repressor is needed to resolve this issue. Another unanswered question is how the two ligands for receptors in a heterodimeric complex differentially contribute to coregulator exchange. Much evidence suggests that there is allosteric communication between receptor partners [61]. Finally, we must remain open-minded with regard to how these structural and dynamic properties are manifested in living cells, where many endogenous coregulators are present and the receptors recruit large multisubunit complexes and bind to DNA in a complex chromatin environment. It is particularly intriguing that receptors bind to their target promoters and recruit coregulator proteins in a cyclical fashion [62]. How these cycles correlate with ligand-induced changes in the receptor remains to be established, but they emphasize the fact that we need to understand not only the dynamics of the receptors themselves, but also their interactions with DNA and co-regulator proteins. Acknowledgements Work in the authors’ laboratories was supported by the Royal Society, the Human Frontier Science Program (HFSP) and a Research Training Network of the European Commission (FP5), by a Research Award from the Boehringer Ingelheim Fund (to L.N.), and by a grant from the
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Hungarian Scientific Research Fund (T034434 to L.N.). L.N. is an International Scholar of the Howard Hughes Medical Institute and an European Molecular Biology Organization (EMBO) Young Investigator.
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30 Sablin, E.P. et al. (2003) Structural basis for ligand-independent activation of the orphan nuclear receptor LRH-1. Mol. Cell 11, 1575– 1585 31 McKenna, N.J. et al. (1999) Nuclear receptor coregulators: cellular and molecular biology. Endocr. Rev. 20, 321 – 344 32 Glass, C.K. and Rosenfeld, M.G. (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121 – 141 33 Chen, J.D. and Evans, R.M. (1995) A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377, 454 – 457 34 Horlein, A.J. et al. (1995) Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377, 397 – 404 35 Sande, S. and Privalsky, M.L. (1996) Identification of TRACs (T3 receptor-associating cofactors), a family of cofactors that associate with, and modulate the activity of, nuclear hormone receptors. Mol. Endocrinol. 10, 813– 825 36 Onate, S.A. et al. (1995) Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270, 1354– 1357 37 Chakravarti, D. et al. (1996) Role of CBP/P300 in nuclear receptor signalling. Nature 383, 99 – 103 38 Ogryzko, V.V. et al. (1996) The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953 – 959 39 Chen, H. et al. (1997) Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90, 569 – 580 40 Heery, D.M. et al. (1997) A signature motif in transcriptional coactivators mediates binding to nuclear receptors. Nature 387, 733 – 736 41 Darimont, B.D. et al. (1998) Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 12, 3343– 3356 42 Hu, X. and Lazar, M.A. (1999) The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature 402, 93 – 96 43 Nagy, L. et al. (1999) Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev. 13, 3209– 3216 44 Perissi, V. et al. (1999) Molecular determinants of nuclear receptor– corepressor interaction. Genes Dev. 13, 3198– 3208 45 Webb, P. et al. (2000) The nuclear receptor corepressor (N-CoR) contains three isoleucine motifs (I/LXXII) that serve as receptor interaction domains (IDs). Mol. Endocrinol. 14, 1976 – 1985 46 Marimuthu, A. et al. (2002) TR surfaces and conformations required to bind nuclear receptor corepressor. Mol. Endocrinol. 16, 271– 286 47 Benko, S. et al. (2003) Molecular determinants of the balance between co-repressor and co-activator recruitment to the retinoic acid receptor. J. Biol. Chem. 278, 43797 – 43806 48 Shiau, A.K. et al. (1998) The structural basis of estrogen receptor/ coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95, 927 – 937 49 Xu, H.E. et al. (2002) Structural basis for antagonist-mediated recruitment of nuclear co-repressors by PPARa. Nature 415, 813 – 817 50 Leng, X. et al. (1993) Ligand-dependent conformational changes in thyroid hormone and retinoic acid receptors are potentially enhanced by heterodimerization with retinoic X receptor. J. Steroid Biochem. Mol. Biol. 46, 643 – 661 51 Keidel, S. et al. (1994) Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping. Mol. Cell. Biol. 14, 287– 298 52 Pissios, P. et al. (2000) Dynamic stabilization of nuclear receptor ligand binding domains by hormone or corepressor binding. Mol. Cell 6, 245– 253 53 Johnson, B.A. et al. (2000) Ligand-induced stabilization of PPARg monitored by NMR spectroscopy: implications for nuclear receptor activation. J. Mol. Biol. 298, 187– 194 54 Cronet, P. et al. (2001) Structure of the PPARa and -g ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure 9, 699– 706 55 Gee, A.C. and Katzenellenbogen, J.A. (2001) Probing conformational changes in the estrogen receptor: evidence for a partially unfolded intermediate facilitating ligand binding and release. Mol. Endocrinol. 15, 421 – 428 56 Kallenberger, B.C. et al. (2003) A dynamic mechanism of nuclear receptor activation and its perturbation in a human disease. Nat. Struct. Biol. 10, 136– 140
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57 Tamrazi, A. et al. (2003) Molecular sensors of estrogen receptor conformations and dynamics. Mol. Endocrinol. 17, 2593– 2602 58 Germain, P. et al. (2002) Co-regulator recruitment and the mechanism of retinoic acid receptor synergy. Nature 415, 187 – 192 59 Stehlin-Gaon, C. et al. (2003) All-trans retinoic acid is a ligand for the orphan nuclear receptor RORb. Nat. Struct. Biol. 10, 820 – 825 60 Forman, B.M. et al. (1998) Androstane metabolites bind to and deactivate the nuclear receptor CAR-b. Nature 395, 612 – 615 61 Nettles, K.W. et al. (2004) Allosteric control of ligand selectivity between estrogen receptors a and b: implications for other nuclear receptors. Mol. Cell 13, 317 – 327 62 Metivier, R. et al. (2003) Estrogen receptor-a directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115, 751 – 763 63 Pike, A.C. et al. (1999) Structure of the ligand-binding domain of oestrogen receptor b in the presence of a partial agonist and a full antagonist. EMBO J. 18, 4608– 4618 64 Black, B.E. et al. (2001) DNA binding domains in diverse nuclear
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receptors function as nuclear export signals. Curr. Biol. 11, 1749 – 1758 Gronemeyer, H. and Moras, D. (1995) Nuclear receptors. How to finger DNA. Nature 375, 190 – 191 Schwabe, J.W. and Rhodes, D. (1991) Beyond zinc fingers: steroid hormone receptors have a novel structural motif for DNA recognition. Trends Biochem. Sci. 16, 291 – 296 Khorasanizadeh, S. and Rastinejad, F. (2001) Nuclear-receptor interactions on DNA-response elements. Trends Biochem. Sci. 26, 384– 390 Schwabe, J.W. and Teichmann, S.A. (2004) Nuclear receptors: the evolution of diversity. Sci. STKE 2004, PE4 Moore, L.B. et al. (2000) Orphan nuclear receptors constitutive androstane receptor and pregnane X receptor share xenobiotic and steroid ligands. J. Biol. Chem. 275, 15122 – 15127 Svensson, S. et al. (2003) Crystal structure of the heterodimeric complex of LXRa and RXRb ligand-binding domains in a fully agonistic conformation. EMBO J. 22, 4625 – 4633
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ’perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information. www.sciencedirect.com
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Mechanism of the nuclear receptor molecular switch Laszlo Nagy1 and John W.R. Schwabe2 1
Department of Biochemistry and Molecular Biology, Research Center for Molecular Medicine, University of Debrecen, Medical and Health Science Center, Nagyerdei Ko¨ru´t 98, Debrecen H-4012, Hungary 2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Nuclear receptors are central to the regulation of development, endocrine signalling and metabolism. The transcriptional activity of many receptors is controlled through the binding of small, fat-soluble molecules to the ligand-binding domain. In most cases, ligand binding turns the receptors into potent activators of transcription. This switch involves the exchange of co-regulator proteins that mediate transcriptional regulation. Structural and biochemical studies have together revealed the mechanism of action of this ligand-induced molecular switch, in which changes in the dynamic behaviour of the receptor play a key role. This remarkable dynamic mechanism has facilitated the evolution of a family of nuclear receptors with highly diverse ligand recognition and signalling properties. The regulation of gene expression requires the ability to switch between two opposing, mutually exclusive or redundant processes. This is essential for the regulation of growth, development and homeostasis in complex multicellular organisms, which use hormones and metabolites to act as messengers that signal the status of the organism as a whole. Fat-soluble hormones and intermediary metabolites are particularly well suited to mediate intercellular communication because, unlike water-soluble peptide hormones, they can pass freely through the lipid bilayer of the cell membrane [1]. Such molecules have been exploited by nature to function as regulatory ligands for a major class of transcriptional regulators called nuclear receptors. These nuclear receptors are special in that they deliver hormonal or metabolic signals directly to the genome by activating or repressing gene expression. In principle, this regulatory system does not require – although it can interact with – additional second messenger systems or protein cascades to exert its effects on transcription. Nuclear receptors were first identified nearly 40 years ago as intracellular receptors for some steroids [2,3]. More than 20 years passed, however, before it became apparent that these steroid receptors are part of a superfamily of metazoan transcription factors that evolved before the divergence of vertebrates and invertebrates and that share a common domain structure consisting of separate DNAand ligand-binding domains [4– 6] (Box 1). The 48 human Corresponding authors: Laszlo Nagy ([email protected]), John W.R. Schwabe ([email protected]). Available online 6 May 2004
members of this family include both receptors for which ligands are known and ‘orphan receptors’ for which there are, as yet, no known ligands [7]. As our understanding of nuclear receptors has grown, it has become increasingly evident that nuclear receptors form a very diverse family in terms of both physiological role and molecular action. The classical view derived from endocrine receptors is that they are each activated by a unique high-affinity ligand with a dissociation constant in the nanomolar range. The best examples of these receptors are members of the steroid hormone receptor family such as the oestrogen receptor (ER) and the androgen receptor. More recently, so-called ‘metabolic receptors’ have been identified that are activated by abundant but low-affinity ligands with dissociation constants in the micromolar range. For example, peroxisome proliferator-activated receptors (PPARs) are activated by various fatty acids, eicosanoids and prostanoids, and liver X receptors (LXRs) are activated by oxysterols [8]. It has also emerged that Box 1. Nuclear receptor domains Nearly all nuclear receptors have two conserved domains (Figure I): a DNA-binding domain (DBD) and C-terminal ligand-binding domain (LBD). A few receptors lacking a DBD have been identified, however, such as the orphan nuclear receptor short heterodimer partner (SHP). Owing to the constraints of maintaining structure and function in a small domain, the DBD is very highly conserved. At its carboxyl terminus is a nuclear localization signal required for nuclear entry; it has been also proposed that a nuclear export signal is located within the DBD itself [64]. The DBD contains two zinc-binding motifs that maintain the domain architecture (reviewed in Refs [65,66]) and have evolved together as a single functional unit. As a result of extensive structural and biochemical investigations, the mechanisms of targetsite recognition by nuclear receptor DBDs are well understood (reviewed in Ref. [67]). Nuclear receptor LBDs are more diverse and are discussed more extensively in the main text. The LBD is primarily helical and has a partly rigid, partly mobile character that might have facilitated rapid evolution [68].
AF-1 <----------------> N–
NES NLS ^ ^ DBD
AF-2 <------------------------> –C LBD Ti BS
Figure I. Common domain structure of nuclear hormone receptors. The N and C termini are indicated. Abbreviations: AF-1, activation function 1 (ligand independent); AF-2, activation function 2 (ligand dependent); DBD, DNA-binding domain; LBD, ligand-binding domain; NES, nuclear export signal; NLS, nuclear localization signal.
www.sciencedirect.com 0968-0004/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2004.04.006
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nuclear receptors can both activate and repress transcription. The role of the ligands can be to activate inactive or repressed receptors, to deactivate or superactivate constitutively active receptors, or, in a few cases, to serve even as non-regulatory structural co-regulators. Finally, it has emerged recently that some receptors apparently lack a ligand binding cavity and therefore might be regulated by other means. The diverse developmental and physiological roles of nuclear receptors are perhaps best illustrated by two examples: the retinoid X receptor (RXR) and the retinoic acid receptor (RAR), which are activated by metabolites of vitamin A and play a central role in the regulation of developmental pathways by through the induction of crucial embryonic Hox genes [9,10]. In a different biological setting, the receptors for some fatty acids (PPARs), bile acids [farnesoid X receptor (FXR); also known as bile acid receptor (BAR)] or cholesterol metabolites (LXRs) regulate complete metabolic pathways such as the uptake, oxidation, storage and intracellular processing of extracellular lipids (reviewed in Ref. [8]). In this way, the metabolic state of the organism programmes the metabolic pathways of a cell through specific nuclear receptors. Because of their diverse and important biological roles, nuclear receptors are primary targets of drug discovery. Furthermore, several receptors are associated with human diseases: for example, RARs are associated with types of leukaemia [11], the ER with breast cancer growth [12], glucocorticoid receptors with inflammation control [13], and PPARs with metabolic diseases such as diabetes [14]. It is evident that elucidating the mechanisms of nuclear receptor activation has widespread implications for our understanding of biological regulation, as well as for efforts to exploit nuclear receptors as drug targets. It is therefore highly significant that the mechanism through which nuclear receptor ligands facilitate the switch from an inactive or repressive condition to an active state has recently become clear through a combination of structural and biochemical techniques. In this review, we describe the studies that have led to our present understanding of the mechanism of the nuclear receptor switch. Specific recognition of a small-molecule ligand There are now numerous structures of nuclear receptor ligand-binding domains (LBDs) bound to small molecules [15]. Such molecules include the natural ligands of the receptor and synthetic agonists and antagonists, as well as adventitiously bound molecules that resemble the natural ligands. By contrast, there are rather few structures of an LBD in which there is no small molecule bound. The first thing to note from the structures is that all of the LBDs share a common, primarily helical, structural scaffold that forms a single protein domain [16]. This domain can be described as a three-layer a-helical sandwich and can be divided into two halves (Figure 1a). In the lower half of the domain there is no central helical layer. Instead, there is a large non-polar cavity in which the various ligands bind. The back and the front of this cavity are sealed by, respectively, a two-stranded b-sheet and the carboxy (C)-terminal helix of the receptor (termed www.sciencedirect.com
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helix 12 on the basis of the structure of apo-RXR [17]). Within this common basic framework, there are several areas where the structure can differ according to the receptor, in particular, the region between helices 1 and 3. Nuclear receptor ligands share in common a mainly non-polar character; however, they vary greatly in size and detailed stereochemistry (Box 2). This variation constitutes the recognition challenge faced by the various LBDs. An important issue, therefore, is how this conserved structural domain can adapt to fit such a wide range of ligands. As one might expect, the diversity in ligand size and shape is matched by a similar diversity in the LBDs themselves. The interior surface of the ligand-binding pockets is mostly made up from non-polar amino acids and thereby matches the character of the various ligands. Specificity is achieved through a limited number of stereo-specific polar contacts and the actual shape of the ligand-binding cavity. It is notable that the receptors that bind with high affinity to a specific ligand, such as the thyroid hormone receptor (TR) and ER, have small ligandbinding cavities [18,19]. Receptors that are apparently more promiscuous and can bind numerous ligands but with lower affinity, such as PPARg and the pregnane X receptor (PXR), have much larger ligand-binding cavities that can accommodate the various ligands in different orientations [20– 23]. It is remarkable that this common structural framework can generate such a vast array of shapes and chemistries of the ligand-binding pocket. The volume of ˚3 this pocket ranges from zero to more than 1000 A (Figure 1b). The larger cavities, such as those in PXR, Ultraspiracle and PPARg, are created by an opening up of the helical framework [20 – 22,24,25]. By contrast, the would-be cavities in the orphan nuclear receptors Nurr1 [26] and DHR38 [27] are completely filled by bulky hydrophobic side chains and a tightening of the helical framework. This lack of a cavity suggests that these receptors are not regulated by conventional ligand binding and either have constitutive activity or are regulated by another mechanism. As mentioned above, only a few structures of a nuclear receptor LBD without a small-molecule ligand have been determined. The first of these was the structure of apo-RXR [17]. Comparison of this structure with the ligand-bound RAR suggested that receptors undergo a very specific switch between two conformations, which involves a major rearrangement of helices 10/11 and 12, as well as more subtle changes [28]. It has subsequently emerged, however, that apo-RXR might be unusual (perhaps because it forms a tetramer in the absence of ligand [29]), because the structures of several other receptors without bound ligand, including PPARg, PXR and liver receptor homologue-1 (LRH-1), closely resemble the ligand-bound state [20,22,30]. These structures suggest that the mechanism of the molecular switch might be more subtle in the latter receptors than it is in RXR. Co-regulators for recruiting large complexes A chief goal of the nuclear receptor field has been to identify the mechanisms and proteins involved in mediating the activation or repression of the receptor itself: in other
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H1
90o
H10/11 H12 H3 (b)
Nurr1 (0 Å3)
TRβ (~600 Å3)
PXR (~1100 Å3) Ti BS
Figure 1. Structural framework of nuclear receptor ligand-binding domains (LBDs). (a) The LBD is formed from a three-layer a-helical sandwich. The layers are shown in blue, yellow and red. In the lower portion of the structure the central helical layer is absent, creating a mainly non-polar cavity in which the ligand (green) binds. Helix 12 and a b-strand (magenta) close the front and back of the ligand-binding cavity, as can be seen on the left. The structure shown is that of the retinoic acid receptor-g (RARg) bound to all-trans retinoic acid (PDB code 2LBD) [28]. (b) Structures of the LBD of Nurr1, the thyroid hormone receptor-b (TRb) and the pregnane X receptor (PXR; PDB codes 1OVL, 1BSX and 1ILG, respectively) [18,22,26]. The ligand-binding cavity is illustrated by a blue mesh: Nurr1 has no cavity; the cavities in TRb and PXR are about 600 and 1100 A˚3, respectively. Note that the size and position of the cavity is dependent upon the extent of opening up of the helices in the lower portion of the structure.
words, how does ligand binding translate into regulation of transcription? This question remains to be answered in full but, at least from the point of view of the receptors, we now have a good understanding of the next step in the signalling process. This progress has been the result of the identification of proteins that interact specifically with either ligand-bound or ligand-free nuclear receptors (reviewed in Refs [31,32]). These proteins are called either co-activator or corepressor proteins, depending on the transcriptional outcome. In general, co-repressors bind to ligand-free nuclear receptors [33 – 35] and are displaced by activating ligands. (Note that some antagonists also displace co-repressors, others are compatible with co-repressor binding, and the so-called ‘inverse agonists’ actually enhance co-repressor binding.) After ligand-binding and displacement of corepressor, co-activator proteins associate with the receptor LBD [36 –39]. After co-regulator proteins were identified, the race was on to establish which part of the co-regulator interacts with the receptor and to identify the interaction surface on the receptor. What has emerged from these studies is a comparatively straightforward mechanism of co-regulator selection. Many co-activators and co-repressors contain multiple, short, receptor interaction motifs, the sequences of which can be generalized as LxxLL in co-activators www.sciencedirect.com
[40,41] and LxxxIxxx[I/L] in co-repressors [42 – 45]. Mutational studies to map the region on the surface of the LBD that interacts with co-activators and co-repressors yielded what seemed at first to be a rather surprising result: that is, co-repressors and co-activators interact with largely overlapping surfaces on the LBD [42 – 47]. Of course, such overlap provides the advantage that binding of the two factors is mutually exclusive and that the only thing that the receptor has to do is to choose which factor can bind. This choice of co-regulator seems to be the switch – in its simplest interpretation – that is controlled by ligand binding. Structural studies of receptor complexes with coactivator peptides have shown that the LxxLL co-activator motif adopts a helical structure on binding to a hydrophobic groove on the surface of the LBD [20,41,48] (Figure 2). The peptide makes contacts with helices 3, 4 and 12. There is a particularly important charge clamp, formed by a lysine and glutamate in helices 3 and 12, respectively, that interacts with the helix dipole. From these structures, it is evident that a particular position of helix 12 is essential to support co-activator binding. It is therefore especially striking that structures of antagonistbound ER show that the inhibitory ligand not only causes helix 12 to be displaced from the position required for co-activator binding, but also itself occupies the
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Box 2. The diversity of nuclear receptor ligands Nuclear receptors can be activated by various fat-soluble molecules, including ‘classical’ steroid hormones such as oestradiol for the oestrogen receptor (ER), cortisol for the glucocorticoid receptor, progestin for the progesterone receptor and testosterone for the androgen receptor. All of these ligands are the products of wellcharacterized steroid hormone biosynthetic pathways. Some vitamins also exert their effects through nuclear receptors. Vitamin-A-derived all-trans retinoic acid and 9-cis retinoic acid function through the retinoic acid (RAR) and retinoid X (RXR) receptors, whereas vitamin D3 activates the vitamin D receptor (VDR). Among intermediary metabolites, an increasing number of mainly low-affinity ligands have been found for previously identified orphan receptors. These metabolites include fatty acids (polyunsaturated fatty acids, oxidized fatty acids), arachidonates and prostanoids for
(a) Chenodeoxycholate
peroxisome proliferator-activated receptors (PPARs), oxysterols for liver X receptors (LXRs) and bile acids for the farnesoid X receptor [FXR; also known as bile acid receptor (BAR)]8. Some receptors, such as pregnane X receptor (PXR), steroid and xenobiotic receptor (SXR), and constitutive androstane receptor (CAR), are even activated by xenobiotics [69]. How xenobiotics can exert evolutionary pressure on these receptors is particularly intriguing. The ability of nuclear receptors to bind to different, structurally distinct ligands (Figure I) is reflected in the structure of their ligandbinding domain (LBD). Unlike classical hormone receptors with tight-fitting LBDs (such as ER and RAR), PPARs and LXRs have large cavities that are only partially filled by ligand [20,70], which explains how more than one type of molecule can regulate their activity.
(c) All-trans retinoic acid
(b) 17β-oestradiol
(d) Rifampicin CH3
CH3
OH COOH
OH
CH3
O
O OH CH3
COOH
O
CH3
OH
CH3 NH
CH3
N
H3CO N OH
OH
N
O
OH
OH
O O CH3
Ti BS
Figure I. Chemical structures of nuclear receptor ligands. (a) The bile acid receptor ligand, chenodeoxycholate (a primary bile acid and a metabolite of cholesterol metabolism). (b) The oestrogen receptor ligand, 17b-oestradiol (a female sex hormone). (c) The retinoic acid receptor ligand, all-trans retinoic acid (a derivative of vitamin A and a potent morphogen). (d) The xenobiotic pregnane X receptor ligand, rifampicin (an antibiotic used in the treatment of tuberculosis).
co-activator-binding groove between helices 3 and 4 [19,48] (Figure 2). This has led to the suggestion that co-repressor peptides might bind to the surface of the LBD in a fashion very similar to that of the coactivators, but that the longer motif might form an extra helical turn such that it does not require helix 12 to be in the active position [42 – 44]. So far, unfortunately, there have been no structures of complexes between ligand-free receptors and co-repressor peptides; however, the structure of antagonist-bound PPARa with a co-repressor peptide has been determined [49]. This structure shows, as predicted from the mapping studies, that the co-repressor peptide does indeed bind in the same hydrophobic groove as the co-activator, with helix 12 displaced from the active position. LBDs as molecular sensors The structural studies discussed above have revealed much of the basis of specific ligand recognition and the nature of the various co-regulator complexes. There is a difference, however, between understanding the various structures and understanding how the molecular switch functions – in other words, the mechanism through which the ligands cause a switch in the choice of co-regulator. The problem is that the structures do not provide a clear explanation for why, in the ligand-free receptor, helix 12 cannot adopt the active position or lead to the recruitment of co-activator proteins. Indeed, in several of the ligandfree structures helix 12 does in fact occupy the active www.sciencedirect.com
position, raising the question of how co-repressor proteins are able to bind to apo-receptors [20,22]. Interestingly, in the complex of PPARa and co-repressor, a synthetic antagonist prevents helix 12 from adopting the active conformation, and thus facilitates co-repressor binding and can be considered an inverse agonist [49]. In summary, it is clear that by themselves the structural studies do not fully clarify the mechanism of the nuclear receptor molecular switch. Fortunately, however, crystallographic studies of nuclear receptors have been complemented by other biochemical and biophysical studies, which have proved to be more fruitful in terms of clarifying the differences between ligand-bound and ligand-free receptors. Proteolytic sensitivity assays show that apo-LBDs are generally more sensitive to proteolysis [50,51]. Gel-filtration analyses suggest that ligand binding slightly reduces the apparent size of the LBD. Thermal denaturation assays, monitored by either circular dichroism or differential scanning calorimetry, show a marked increase in the melting temperature of the LBD on ligand binding [23]. Finally, dynamic stabilization assays investigating the interaction between the isolated helix 1 and the remainder of the LBD show that there is a significant stabilization of this interaction on the addition of ligand (and also co-regulators) [52]. Taken together, these results strongly suggest that ligand binding stabilizes the receptor LBD, resulting in a more compact and rigid structure.
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(a)
ERα + agonist
ERα + agonist + CoA
ERα + antagonist
ERβ + antagonist
(b)
Apo-PPARγ 1
Apo-PPARγ 2
PPARα + agonist + CoA
PPARα + antagonist+ CoR
Figure 2. Effect of ligands and co-regulators on the position of helix 12. The top row of structures shows the oestrogen receptor (ER) bound to agonists and antagonists (PDB codes 1ERE, 1ERR, 1GWR and 1QKM) [19,63]. Note that in the antagonist-bound structures helix 12 adopts a position very similar to that of the co-activator in the agonist-bound structure. The bottom row of structures shows peroxisome proliferator-activated receptors (PPARs) in the absence of ligand (left two structures; PDB code 1PRG), bound to agonist and co-activator (PDB code 2PRG) and to antagonist and co-repressor (PDB code 1KKQ) [20,49]. Note that in the absence of ligand, helix 12 can adopt both the active position and an alternative position. In the presence of antagonist and co-repressor, helix 12 unravels. ERs and PPARs are shown in light blue and green, respectively; helix 12 in yellow; agonists and antagonists in magenta and cyan, respectively; and co-activator and co-repressor peptides in red and dark blue, respectively.
This conclusion is complemented by two further experimental observations. First, the crystal structure of apo-PPAR-g shows a striking distribution of crystallographic temperature factors [20] (Figure 3a), which seems to suggest that the lower portion of the LBD, as seen in Figure 3a, is less rigid than the upper portion. This idea is further supported by NMR studies of apo- and holoPPARg and PPARa, which found that essentially the same region of the ligand-free LBD shows exchange broadening on a millisecond timescale [53,54], suggesting that there is significant conformational mobility but on a timescale slower than the overall tumbling of the protein. Altogether, these findings fit with suggestions that the lower portion of the receptor is rather dynamic and might have some of the properties of a molten globule [55]. The dynamic properties of the apo- and holo-receptors are thus distinct. In itself, however, this difference does not explain how the choice of co-regulator binding is controlled. The key to the binding choice seems to be the dynamic behaviour of helix 12 of the receptor. Fluorescence anisotropy techniques show that helix 12 is very dynamic in apo-PPARg, with motions on the scale of a few nanoseconds, which suggests that it has mobility independent of the rest of the LBD [56]. On ligand binding, helix 12 shows significantly slower dynamics, suggesting that it is bound to the surface of the LBD, presumably in the active conformation [56]. Similar studies of the ER www.sciencedirect.com
containing a fluorescent label near the C-terminus of helix 11 support the finding that ligand binding has a profound effect on the fast motions of this region of the LBD [57]. Ligand binding probably stabilizes helix 12 in the active conformation in two ways. First, in PPARg, as in many other receptors, the ligand itself makes direct contact with residues in helix 12, thereby promoting the active conformation. Second, the ligand globally stabilizes the lower half of the LBD, and this in itself will favour helix 12 stably adopting the active conformation. Of course, both of these mechanisms also promote the binding of co-activator peptides. In the absence of ligand, the larger co-repressor interaction motif can bind to the LBD and partially stabilize the ligand-free conformation, as observed in the dynamic assembly assay [52]. It is also important to note that the relative amount of co-repressor and co-activator has been shown to influence the equilibrium between the active and inactive conformations [58]. In summary, it is clear that the ligand-free LBD of PPARg is partly rigid and partly mobile. Helix 12 of the receptor is especially mobile, showing fast, independent, segmental motion [56]. Ligand binding globally stabilizes the LBD and, through both this and direct contacts, stabilizes helix 12 in the active conformation, which in turn promotes co-activator binding. This global stabilization can be perhaps considered as the switch mechanism,
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(a) Upper portion Tightly packed, three-layer helical sandwich Lower crystallographic temperature factors Sharp peaks in NMR spectra Forms rigid support for the apo-LBD Helix 12 Short C-terminal helix of LBD Fast segmental mobility (nanosecond timescale) Dynamic behaviour sensitive to different classes of ligand Lower portion Helices framing ligand-binding cavity Higher crystallographic temperature factors Conformationally labile (millisecond timescale) Dynamic character analogous to molten globule (b) Basal transcriptional activity Helix 12 mobility and LBD dynamics
RARα
PPARγ
LRH-1 Ti BS
Figure 3. Nuclear receptor dynamics and activity. (a) Structure of a ligand-free peroxisome proliferator-activated receptor (PPARg; PDB code 1PRG) [20] coloured according to crystallographic temperature factors and annotated according to our understanding of the dynamic properties of this molecule [53,56]. (b) Different nuclear receptors are likely to show different degrees of dynamic lability that correlate with their basal transcriptional activity. For example, liver receptor homologue-1 (LRH-1) has constitutive activity and is stable enough to be crystallized in the absence of ligand; retinoic acid receptor-a (RARa) is a repressor in the absence of ligand and crystallization attempts without ligand have been unsuccessful, which is consistent with the idea that the ligand-binding domain (LBD) is conformationally mobile; peroxisome proliferatoractivated receptor-g (PPARg) adopts an intermediate position between these extremes.
with helix 12 acting as the sensor through which the state of the receptor is read by co-regulators. The next question is whether this mechanism, established for PPARg, applies to other nuclear receptors, especially because nuclear receptors are rather diverse in their mechanism of action. The available evidence suggests that nuclear receptors show a great range in dynamic behaviour that correlates rather well with their level of basal activity. It seems likely, therefore, that there is a stability scale on which the nuclear receptors adopt different positions (Figure 3b). The LBDs of LRH-1 and Nurr1 are constitutively active. Both of these have been crystallized in the absence of ligand, revealing stable structures with helix 12 in the active conformation [26,30]. By contrast, ligand-free TR and RAR are strong repressors, have proved so far to be refractory to crystallization efforts, and show rather low melting temperatures in the absence of ligand. Thus, TR and RAR probably sit at the more dynamic end of the scale. PPARg sits between these two extremes, with a rather high basal activity but with the ability to be activated by ligand. Concluding remarks The realization that the dynamic behaviour of the LBD is key to the mechanism of the molecular switch explains some hitherto puzzling observations about nuclear receptors. In particular, it explains how selective modulators can fine-tune the activity of the receptor [57]. Hence, the switch is not like a regular on – off light switch, but is more like a dimmer switch. This in turn explains why some receptors, such as RAR-related orphan receptor-b (RORb) and constitutive androstane receptor (CAR), seem to www.sciencedirect.com
function in the reverse direction, with ligands serving as deactivators [59,60]. But several key questions remain unanswered. It is still not fully clear how co-repressors bind to apo-receptors. The only available structure – that of PPARa bound to a SMRT (silencing mediator of RAR and TR) peptide – is in the presence of an antagonist ligand, which is likely to stabilize the LBD structure. The structure of a ligandfree repressing receptor, such as RAR, TR or LXR, bound to co-repressor is needed to resolve this issue. Another unanswered question is how the two ligands for receptors in a heterodimeric complex differentially contribute to coregulator exchange. Much evidence suggests that there is allosteric communication between receptor partners [61]. Finally, we must remain open-minded with regard to how these structural and dynamic properties are manifested in living cells, where many endogenous coregulators are present and the receptors recruit large multisubunit complexes and bind to DNA in a complex chromatin environment. It is particularly intriguing that receptors bind to their target promoters and recruit coregulator proteins in a cyclical fashion [62]. How these cycles correlate with ligand-induced changes in the receptor remains to be established, but they emphasize the fact that we need to understand not only the dynamics of the receptors themselves, but also their interactions with DNA and co-regulator proteins. Acknowledgements Work in the authors’ laboratories was supported by the Royal Society, the Human Frontier Science Program (HFSP) and a Research Training Network of the European Commission (FP5), by a Research Award from the Boehringer Ingelheim Fund (to L.N.), and by a grant from the
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Hungarian Scientific Research Fund (T034434 to L.N.). L.N. is an International Scholar of the Howard Hughes Medical Institute and an European Molecular Biology Organization (EMBO) Young Investigator.
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