Looking at nuclear receptors from a new angle

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Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce

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At the Cutting Edge

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Looking at nuclear receptors from a new angle

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Q1

Christine Helsen, Frank Claessens ⇑

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Laboratory of Molecular Endocrinology, Department of Cellular and Molecular Medicine, KU Leuven, O&N1, Herestraat 49, 3000 Leuven, Belgium

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a r t i c l e

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i n f o

Article history: Received 16 July 2013 Received in revised form 5 September 2013 Accepted 6 September 2013 Available online xxxx

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Keywords: Nuclear receptor Steroid receptor DNA binding Domain communication

a b s t r a c t While the structures of the DNA- and ligand-binding domains of many nuclear receptors have been determined in great detail; the mechanisms by which these domains interact and possibly ‘communicate’ is still under debate. The first crystal structures of receptor dimers bound to ligand, DNA and coactivator peptides provided new insights in this matter. The observed binding modes revealed exciting new interaction surfaces between the different nuclear receptor domains. Such interfaces are proposed to be the route through which allosteric signals from the DNA are passed on to the ligand-binding domain and the activating functions of the receptor. The structural determinations of DNA-bound receptor dimers in solution, however, revealed an extended structure of the receptors. Here, we discuss these apparent contradictory structural data and their possible implications for the functioning of nuclear receptors. Ó 2013 Published by Elsevier Ireland Ltd.

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Contents

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1. 2. 3.

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4. 5. 6. 7. 8.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA binding by nuclear receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Domain organization of nuclear receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The ligand-binding domain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The DNA-binding domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. The hinge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. The NTD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Allosteric communication in nuclear receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural data supporting domain communications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The open/extended conformation of DNA-bound receptor dimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How to reconcile the new data? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural extrapolation to other nuclear receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: AF1, activation function 1; AF2, activation function 2; AR, androgen receptor; CTE, carboxyterminal extension; DR, direct repeat; DBD, DNA binding domain; EM, electron-microscopy; ER, estrogen receptor; ERR2, estrogenrelated receptor 2; FRET, fluorescence resonance energy transfer; GR, glucocorticoid receptor; HNF-4, hepatocyte nuclear factor 4; IR, inverted repeat; LBD, ligand binding domain; MR, mineralocorticoid receptor; NR, nuclear receptor; NTD, aminoterminal domain; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; SANS, small-angle neutron scattering; SAXS, small-angle X-ray scattering; SF1, steroidogenic factor 1; TR, thyroid receptor; VDR, vitamin D receptor. ⇑ Corresponding author. Address: Laboratory of Molecular Endocrinology, Department of Cellular and Molecular Medicine, Onderwijs & Navorsing 1, Box 901, Herestraat 49, 3000 Leuven, Belgium. Tel.: +32 16 330253; fax: +32 16 330735. E-mail address: [email protected] (F. Claessens).

1. Introduction

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Nuclear receptors (NRs) play a crucial role in many physiological processes such as reproduction, metabolism, inflammation, immunity and lipid signaling (Hollman et al., 2012; Lamers et al., 2012; Pascual-Garcia and Valledor, 2012; Verhoeven et al., 2010). As much as 48 human NRs have been identified thus far (Xiao et al., 2013). Nuclear receptors are activated by their cognate ligands or other signals and function as transcription factors. Upon activation, the NR will bind to a specific DNA sequence, named the

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0303-7207/$ - see front matter Ó 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.mce.2013.09.009

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response element, located in the regulatory regions of their target genes. When binding to the promoter or enhancer regions of the target genes, the receptor will affect transcription by recruiting specific co-regulators and components of the transcription initiation complex or RNA polymerase II (Acevedo and Kraus, 2004). Detailed insight in the structure–function relationship of NRs originates from crystallographic studies on the two most conserved domains: the DNA binding domain (DBD) and the ligand binding domain (LBD). The aminoterminal domains of NRs are highly variable in length and in sequence. Structural studies indicate they are flexible and most likely intrinsically disordered (Khan et al., 2011; Kumar and Litwack, 2009; Kumar and Thompson, 2012). The hinge regions which connect the DNA- with the ligand-binding domains are the least conserved between the members of the NR family and their structures are poorly understood. Crystallographic data for receptor dimers binding to DNA as well as coactivator peptides have now been reported for the PPARc (peroxisome proliferator-activated receptor)–RXRa (retinoid X receptor) heterodimer and for the HNF-4a (hepatocyte nuclear factor 4) homodimer (Chandra et al., 2008, 2013). Solution structures of several receptor heterodimers were obtained via small-angle Xray scattering (SAXS), small-angle neutron scattering (SANS) and electron-microscopy (EM) (Rochel et al., 2011). Here, we focus on how these different structures can be reconciled and what novel insights and questions are evoked by them, with regard to steroid receptor action.

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2. DNA binding by nuclear receptors

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Extensive study of DNA binding by NRs has shown that the global composition of the DNA response element determines which NR can bind to it. Response elements are typically composed of

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Table 1 DNA binding by the NR family.

I. Steroid receptors

NR

Consensus RE

Dimerization

Configuration

AR, PR

50 -AGAACA30 50 -AGAACA30 50 -AGGTCA30

Homodimer

IR3, DR3

Homodimer

IR3

Homodimer

IR3

50 -AGGTCA30

Homodimer

IR0

Heterodimer

Homodimer

DR1 DR2 DR5 DR3

Heterodimer Heterodimer

DR3 DR1

Monomer

Half-site

Homodimer Heterodimer Homodimer

DR4, IP6, P0 DR4 DR1

Heterodimer

DR1 DR2 DR3 DR4 DR5

GR, MR ER II. Non-steroid receptors

RAR

VDR

PPAR TR

RXR

III. Orphan receptors

Nurr77 SF1, ERR2

50 -AGGTCA30 50 -AGGTCA30 50 -AGGTCA30

50 -AGGTCA30

50 -AAA AGGTCA-30 50 -TCA AGGTCA-30

Monomer Monomer

Extended half-site Extended half-site

two hexameric sequence organized as a direct, inverted or everted repeat. Each hexameric sequence or half-site is recognized by a receptor (Roemer et al., 2006). The half-sites are usually separated from each other by a spacer with variable length. Less common are response elements that consist of only 1 hexameric sequence which is recognized by a NR in monomeric binding mode. The exact composition and hence recognition by the correct NR is dependent on orientation and sequence of the hexamer and on the spacer length. Steroid receptors prefer the 50 -AGAACA-30 -like motifs while non-steroid receptors and the ER bind to the 50 -AGGTCA-30 -like motifs. The specific DNA binding properties of each receptor will enable or disable binding to a certain response element. Properties such as the flexibility and the length of the hinge, the specific recognition of the half-site and the strength of intermolecular dimerization via the DBDs and LBDs allow adaptation for the correct positioning of the receptors. Roughly, the NRs can be subdivided into three groups based on their DNA binding characteristics: receptors that homodimerize, receptors that heterodimerize with one of the retinoid X receptors (RXRs) and receptors that bind as a monomer (Table 1). The first group of homodimeric receptors consists of the steroid receptors: the estrogen receptor (ER), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), the progestagen receptor (PR) and the androgen receptor (AR). They can homodimerize on an inverted repeat of 50 -AGAACA-30 -like motifs with a 3-nucleotide spacer (IR3) (50 -AGGTCA-30 for the ER) (Table 1). The AR and the PR can also bind to 3-nucleotide spaced direct repeats of the same hexamer (DR3) (Denayer et al., 2010; Kerkhofs et al., 2012). The second group of receptors comprises the receptors that can heterodimerize with RXR, although some of them can also homodimerize. They recognize direct repeats of 50 -AGGTCA-30 -like motifs with receptor-specific spacer lengths (Table 1) (Rastinejad et al., 1995). Monomeric DNA binders, such as Nur77 (Meinke and Sigler, 1999), SF1 (steroidogenic factor 1) (Little et al., 2006) and ERR2 (estrogen receptor-related receptor 2) (Gearhart et al., 2003), are known to extend the DBD-DNA interface outside the major groove of the DNA. Additional contacts are formed between the CTE of the orphan receptor and the minor groove of the DNA upstream of the hexameric consensus sequence (Table 1). Alternatively, receptors are recruited to DNA indirectly via other sequence-specific transcription factors (Heldring et al., 2011; Sahu et al., 2011), but this will not be discussed here.

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3. Domain organization of nuclear receptors

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3.1. The ligand-binding domain

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The 3-dimensional structure of the LBD consists of 12 a-helices in antiparallel sandwich-like arrangement (Wurtz et al., 1996). A comparison of the structures in absence and in presence of hormone led to the ‘mouse trap’ hypothesis (Parker and White, 1996) which was later confirmed by the structures of many NRLBDs. When an agonist binds the ligand-binding pocket, helix 12 serves as a lid and covers the ligand-binding pocket. This repositioned helix 12 forms a platform for coactivator binding (Brzozowski et al., 1997; Parker and White, 1996). Co-crystals of LBDs with coactivator peptides that contain the nuclear receptor binding signature motif (Heery et al., 1997) illustrated how helix 12 forms the activation function 2 (AF2) surface first proposed by Cavailles (Cavailles et al., 1994). Antagonist binding will reposition helix 12 differently and, as a result, the LBD fails to recruit coactivators. The alternative surface can even lead to binding of corepressors which results in a transcriptionally repressed target gene (Heldring et al., 2007). Structures of agonist and antagonist bound LBDs have had a major impact and have provided valuable

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for the design and in silico screening of new synthetic ligands (Bruning et al., 2010; Buzon et al., 2012; Munuganti et al., 2013; Nilsson et al., 2011; Voet et al., 2013) or to explain the physiological consequence of receptor mutations (Chandra et al., 2013; Hughes et al., 2012; Massin et al., 2012). For most receptors, AF2 located in the LBD is the main activation function. For other receptors, a second transactivation unit (AF1), located in the aminoterminal domain (NTD), contributes to the transcriptional regulation of gene expression by AF2. The LBDs of the non-steroid receptors and of the ER also contain a surface required for receptor dimerization. This interface is formed by helix 10, helix 9 and the loop between helix 7 and 8 (Bourguet et al., 1995). Dimerization via the NR-LBDs is said to occur in solution and to facilitate dimerization via the DBDs (Perlmann et al., 1996). While such dimerization via the LBD is well-known for VDR (vitamin D receptor), TR (thyroid receptor), PPAR, RAR (retinoic acid receptor) and RXR, there is no clear evidence that it occurs for GR, MR, PR and AR. Double hybrid analyses e.g. show no evidence for LBD–LBD interactions for the AR (Doesburg et al., 1997). The crystallographic data on the LBD-dimers of GR, MR and PR (PDB ID 1M2Z, 2AAX, 1ZUC) do not show similar protein– protein contacts nor do they reconcile the dimerization surface reported for the heterodimeric nuclear receptors (Bledsoe et al., 2005, 2002; Zhang et al., 2005). Since the AR LBD crystallized as a clear monomer (PDB ID 2AMA) (Pereira de Jesus-Tran et al., 2006) and since the protein–protein contacts reported in GR, PR and MR LBD crystals seem to be crystal packing contacts rather than genuine dimerization surfaces (Brelivet et al., 2012), these receptors do not seem to dimerize via their LBD to enable DNA binding. The presence of a b-strand C-terminally of helix 12 in the AR, GR, MR and PR LBD structures could be a possible explanation for the absence of dimerization, since it covers helix 9, 10–11 and thereby prevents LBD dimerization as known for the non-steroid receptors ((Schoch et al., 2010) and Fig. 1). It is thus uncertain and even unlikely that dimerization of AR, GR, PR and MR LBDs ever occurs.

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3.2. The DNA-binding domain

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The DBD is the most conserved domain among the nuclear receptors. It consists of two zinc finger modules (reviewed in (Helsen et al., 2012b)) that form the core structure. The a-helix in the first zinc finger module enables the sequence–specific interactions with the DNA. The second zinc finger module allows the receptorDBDs to hetero- or homodimerize. The DBD also contains a variable C-terminal extension which either stabilizes DNA-binding by making contact in the minor groove of the DNA or participates in

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AR

protein–protein contacts of the DBD-dimer (Hsu et al., 1998; Roemer et al., 2006; Shaffer and Gewirth, 2002). As mentioned above, the DBD dimerization has to be in sync with the format of the hormone response element. The variations in spacer length and hexamer orientations have to be accommodated by the 2nd zinc finger and the CTE (Richmond and Davey, 2003).

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3.3. The hinge

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The hinge is a flexible region located between the LBD and the DBD. For many NR, the hinge contains a nuclear localization signal and sites for post-translational modifications like phosphorylation, acetylation, methylation and sumoylation (Anbalagan et al., 2012; Clinckemalie et al., 2012). For some receptors, the length of the hinge and not the amino acid sequence seems to matter for proper functioning (Shaffer et al., 2005), while for other receptors mutational studies have indicated that specific residues within the hinge region can have an important impact on nuclear import, DNA binding, receptor stability and intracellular mobility (Daniel et al., 2012; Held et al., 2012; Tanner et al., 2010).

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3.4. The NTD

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The NTD is the most diverse domain among NRs. Neither its length nor its sequence is conserved. The steroid receptors AR, GR, MR and PR have the largest NTD domains, ranging from 400 to 600 amino acids with MR having the largest NTD of 602 aa (Fig. 2) (Yang and Fuller, 2012). The NTDs of the non-steroid receptors are much shorter. The NTD of the vitamin D receptor is, for example, only 24 amino acids long (Campbell et al., 2010). Most NTDs contain an activation function traditionally called AF1. This activation function acts as multiple signal input and output domain integrating signals from different pathways which can converge here, sometimes in cooperation with the signals that modulate the receptors activity via the LBD, the hinge and the DBD. However, the NTD-mediated mechanisms are mostly receptor-specific. For the AR, e.g. over 150 coregulators have been reported to interact to AF1, some of which are known AF2 interacting proteins or complexes (Callewaert et al., 2006; Heemers and Tindall, 2007; Lavery and McEwan, 2005; van de Wijngaart et al., 2012). While in absence of binding partners the NTD is believed to be intrinsically disordered, interaction with their binding partners might induce appropriate folding in the activation functions. In fact, induced folding could be a mechanism to enable interaction with numerous different binding partners (Almlof et al., 1998; Dotzlaw et al., 2002; Warnmark et al., 2001). Unfortunately, the

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ER

H10

VDR

H10 H10

H12

H12

H11

H11

H12

H11

Fig. 1. The LBD structure of AR, ER and VDR. PDB IDs: 2AMA, 2QXS and 3A78. Helix 12 (red) closes off the ligand-binding pocket. The b-sheet carboxyterminal of helix 12 in the AR (blue) is not present in ER and non-steroid receptors. Helix 10 and helix 11 (green) are the most important structural elements for homodimerization of ER and for homo- and heterodimerization of VDR. In AR, the C-terminal b-sheet is shielding this interface potentially preventing LBD dimerization. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Domain composition and domain interactions of nuclear receptors. Steroid receptors contain large NTDs that are capable of interacting with the LBD (N/Cinteraction). LBD-dimerization is best described for the non-steroid receptors. For the estrogen receptor (ER), however, both N/C-interaction and LBD-dimerization have been described.

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structure of the NR-NTDs or even functional subdomains remains largely undiscovered. The protein fragments are present but remain invisible in the recently reported crystals of the holoreceptors (Chandra et al., 2008, 2013), so we rely on more indirect techniques to solve overall NR structures.

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4. Allosteric communication in nuclear receptors

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Nuclear receptors are modular proteins composed of different domains that enable the receptor to fulfill its function as ligandactivated transcription factor (Claessens et al., 2008). Despite the fact that the domains can execute their prime functions of transactivation, DNA and ligand binding in isolation, there is experimental evidence for functional communications between the different domains. For the GR for instance, the lever arm in the DBD is responsible for mediating communication from the response element towards the LBD and vice versa, the binding of ligand in the LBD of the GR differentially influences the binding of the receptor to a series of glucocorticoid response elements (Meijsing et al., 2007, 2009; van Tilborg et al., 2000). Also for the ER, both the DNA sequence of the estrogen response elements and the ligand fine-tune the activity of the ER by regulating DNA-binding by the receptor (Deegan et al., 2011; Margeat et al., 2003). Hydrogen–deuterium exchange was used to study the receptor dynamics of the VDR– RXR heterodimer in response to DNA and ligand binding. This study confirmed the allosteric communication between the DBD

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and LBD transferring the signal from the DNA towards coactivator binding sites and vice versa (Zhang et al., 2011). The DBD has been reported to also influence the NTD-mediated functions. Binding of the AR DBD to a response element will induce structure in the NTD, which was proposed to act as a mechanism to regulate gene expression by enhancing interaction with certain coactivators (Brodie and McEwan, 2005). For the GR DBD, binding of JDP2 induces folding in AF1 leading to improved interaction with CBP and TBP but not SRC-1 (Garza et al., 2011). The NTD of some NRs can also influence the LBD by directly interacting with the hydrophobic coactivator binding groove located at the surface of the LBD. This N/C-interaction has been best characterized in the AR (reviewed in (van Royen et al., 2012)) where it is required for full transcriptional activity on response elements organized as IR3 but not on DR3-organized elements (Callewaert et al., 2003). These N/C-interactions for the AR occur in a spatiotemporal way. Ligand binding first induces an intramolecular bridge. Subsequently, intermolecular N/C interactions occur in the AR homodimer when the receptors are traveling through the nucleus, but once bound to the DNA the N/C interaction is largely lost, thus enabling interactions with coregulators (van Royen et al., 2007). For MR, PR-B and ERa, N/C interactions have also been reported (Kraus et al., 1995; Metivier et al., 2001; Pippal et al., 2009; Takimoto et al., 2003; Tetel et al., 1999). Despite the smaller size of the NTD, N/C interactions have even been suggested for the PPARc (Shao et al., 1998). How the receptor domains communicate, whether it occurs intra- or intermolecular, how these interactions take place, how they are regulated in time and how other proteins or posttranslational modifications are affecting them, remains largely undiscovered. The structural insights on full size receptors or larger fragments are now unveiling the first details.

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5. Structural data supporting domain communications

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The group of Rastinejad reported the first crystal structure of full size nuclear receptors, they observed a closed conformation for the PPARc–RXRa heterodimer (Figs. 3 and 4) (Chandra et al., 2008). The fold and dimerizations of the DBDs and the LBDs are similar to those of isolated DBDs and LBDs reported earlier, and each of the liganded LBDs was bound by one LXXLL coactivator peptide (Connors et al., 2009; Gampe et al., 2000; Holmbeck et al., 1998). Unfortunately, the structure of the NTDs of PPARc or RXRa could not be derived from the crystal structure, presumably due to their high mobility (Chandra et al., 2008). It is, however, the global organization of PPARc and RXRa that revealed new insights on NR dimerization (Fig. 3). The configuration of the PPARc–RXRa heterodimer on its DNA response element is largely asymmetrical, with the RXRa being stretched out, enclosing the LBD of PPARc between its LBD and DBD. This reveals a new, previously unknown interface between PPARc LBD and RXRa DBD. Furthermore, the hinge of PPARc interacts with the 50 upstream region of the DR1 determining the polarity of the PPARc–RXRa binding orientation. The hinge (CTE-region) of RXRa, on the other hand, is involved in the DNA-dependent dimerization of the DBDs in a head-to-tail conformation on the DR1. The relevance of the PPARc LBD–RXRa DBD surface was demonstrated by reduced DNA binding ability after mutation of a residue (Phe 347 Ala) in the human PPARc LBD (Chandra et al., 2008). In short, the PPARc LBD contacts both DBDs to stabilize DNA binding. Moreover, phosphorylation of Ser 273, which is in the DBD–LBD interface in mouse PPARc LBD results in changes in PPARc-dependent gene expression (Choi et al., 2010). Possibly, this phosphorylation acts via the modulation of the DBD–LBD interactions.

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Fig. 3. Available structures of full-size nuclear receptors. The structures of PPAR–RXR heterodimer and the HNF-4a homodimer were determined via crystallography. The structures of PPAR–RXR (DR1), RAR–RXRa (DR1), VDR–RXRa (DR3) and RAR–RXRa (DR5) were obtained via SAXS, SANS and cryo-EM. The closed and open/extended conformation of PPAR–RXR are both depicted. DBD are depicted as triangles, LBDs as circles, coactivator peptides as green cilinders. DRn are response elements composed of a direct repeat of two half-sites spaced by n nucleotides.

A

B

Fig. 4. The two available models of full-size NR dimers via crystallography. (A) The PPAR–RXR dimer on a DR1, PPAR in red and RXR in blue. (B) The HNF-4a homodimer on a DR1, one receptor in yellow, the other in orange. Axial view on the DNA helix. While the DBDs are positioned in a similar way on the DR1 in each model, the position of the LBDs differs substantially between these models. Coactivator peptides are depicted in magenta. DR1 is a response element composed of a direct repeat of two half-sites spaced by 1 nucleotide. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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These observations open up the possibility that a ligand which binds to the LBD or a compound that affects the posttranslational modifications can differentially affect receptor activity on specific DNA response elements and thus selectively regulate gene expression by these receptors (Zhang et al., 2011). Unfortunately due to crystal packing constraints that trap minor conformers within the agonist–antagonist equilibrium, these LBD–DBD communications were not captured in these crystal structures of PPAR–RXR with structurally and functionally distinct PPAR ligands (agonist and antagonists). The different crystal structures (PDB ID: 3DZY, 3DZU and 3E00) even failed to demonstrate the expected ligandinduced differences at the level of helix 12 (Chandra et al., 2008). Vice versa, small variations in the DNA response element will likely affect the binding strength/mode of the receptor complex, however these effects were not studied here since these nuclear receptor complexes were made with consensus half-sites for optimal binding. For the GR, it was demonstrated that addition of an extra nucleotide in the 3-nucleotide spacer of a consensus

response element could not affect the position of the DBD-dimer on its response element due to the stabilization of DNA binding through a large DBD dimerization surface (Luisi et al., 1991). For PPAR–RXR dimer, however, the DBD dimerization surface is only modest (30 Å2) which emphasizes the importance of DBD–DNA contacts and the DNA dependence of this DNA binding mode (Chandra et al., 2008). Therefore, it is likely that the fine molecular contacts in the PPAR–RXR dimer will differ slightly on naturally occurring response elements compared to the binding on consensus DNA response elements. The group of Rastinejad published a second X-ray based NR-dimer structure, this time for HNF-4a which binds as a homodimer to a DR1 element (Figs. 3 and 4)(Chandra et al., 2013). The crystallized fragment consisted of a DBD–LBD fragment, as well as the response element and coactivator peptides. In the asymmetrical homodimer, the DBD of the upstream monomer contacts both LBDs as well as the DBD and hinge region of the downstream partner. The discovery of these interfaces provides insights in

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the effects of two posttranslational modifications: the methylation of an arginine and the phosphorylation of a serine (Chandra et al., 2013). Both modifications affect receptor activity and are located in the DBD–LBD interface. The authors also found strong supporting evidence for the interfaces in the effects of some naturally occurring mutations in HNF-4a which can now be explained only by the fact that they disrupt the DBD–LBD interfaces (Chandra et al., 2013). In conclusion, the structures of the PPAR–RXR and HNF-4a dimers both show important quaternary conformations which can provide logic explanations to earlier biochemical observations of allosteric communications. However, while the DBD-dimers and the LBD-dimers are similar, the quaternary interactions are very different. Indeed, when both structures (PDB IDs: 3DZY and 4IQR) are aligned according to the DBDs, the positions of the LBDs are at different sides of the DNA (Fig. 4). If this variability would hold true for other nuclear receptors, it would lead to an important increase of structural variation.

6. The open/extended conformation of DNA-bound receptor dimers The group of Moras used SAXS, SANS and FRET (Fluorescence resonance energy transfer) to analyze the solution structures of DNA bound full size receptors. They reported an open conformation for several non-steroid receptors (homodimers and heterodimers with RXR; Fig. 3) (Orlov et al., 2012; Osz et al., 2012a,b; Rochel et al., 2011). On the same DNA response element as used for the crystal structure (DR1), the PPAR–RXR heterodimer forms an elongated asymmetric shape with the LBDs well separated from the DBDs by the hinges (Rochel et al., 2011). Although the resolution is not as high as in X-ray data, the mode of DBD and LBD dimerization agrees with the structures previously reported for the isolated domains. The LBD dimer, however, is positioned above the DBD located at the 50 end of the element, with the two hinges in parallel (Fig. 3). The hinge of PPAR is not contacting the DNA upstream of the 50 half-site which is in contrast with the crystal structure. Next to the structure of PPAR–RXR, the solution structures of other RXR heterodimers were also determined: RAR–RXR on DR1, RXR–VDR on DR3 and RXR–RAR on DR5 (Rochel et al., 2011). While the position of the DBDs in these RXR heterodimers depends on the composition of the response element, the position and the orientation of the dimerized LBDs is always very similar and positioned above the upstream DBD (Rochel et al., 2011). The hinge of the receptors seems to play an important role in adapting to differences in spacer lengths and hexamer orientations to still allow the regular DBD and LBD dimerization. The cryo-EM structure of DNA-bound RXR–VDR on a DR3 shows an extended RXR hinge region, while the hinge of VDR contains a rigid CTE helix that determines the topology of the complex (Orlov et al., 2012). Furthermore, the localization of the short NTD of VDR could also be determined in this receptor complex. Interestingly, it interacts with the major groove of the DNA response element, next to the recognition helix of VDR. This could point towards a modulating effect on DNA binding (Orlov et al., 2012). The short NTD of VDR does not contain an AF1 function (Sone et al., 1991), but shortening of the NTD with 3 amino acids, a naturally occurring polymorphism, has been shown to influence the repressive function of the VDR in the absence of ligand (Alimirah et al., 2010). The domain composition of these RXR heterodimers is for the most part comparable to the quaternary structure of the HNF-4a homodimer which also shows both LBDs above the 50 DBD (Chandra et al., 2013). However, the overall domain composition of the HNF-4a crystal structure is more compact with the LBDs

contacting the DBD and not, as was demonstrated for these RXR heterodimers, an extended hinge that keeps the LBDs apart from the DBDs (Rochel et al., 2011).

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Different techniques have led to two apparent contradictory conformations for PPARc–RXRa. Should we assume that the existence of both an extended and a closed conformation for the PPARc–RXRa dimer underlines the general flexibility of the multi-domain NRs? (Brelivet et al., 2012; Chandra et al., 2008; Osz et al., 2012b). Both conformations could indeed support different aspects of receptor functioning. While the open conformation allows coregulators and other proteins to interact with the available surfaces on the DBD, the LBD and the hinge, the closed conformation could serve e.g. to reinforce or stabilize binding of the receptor complex to the response element and/or conducting allosteric signals between these domains, the DNA and interacting complexes. Both reported receptor models could be snapshots of a receptor complex that is in constant equilibrium between several energyfavorable conformations. Both extended and closed receptor conformations provide alternative surfaces that, much like AF2, can be targets for alternative protein–protein interactions that can play a role in processes directly or indirectly linked to transcriptional regulation. It should be noted that even when only the LBD is taken into account, molecular dynamics indicated several flexible surfaces that could allosterically modulate the activity of the receptor (Buzon et al., 2012; Grosdidier et al., 2012). Such transitions will be regulated posttranslationally (cfr. (Chandra et al., 2013)) and/or direct the protein–protein or protein–DNA interactions at the enhancer site. The complex interplay between the different domains of receptors also came to the surface when determining the stoichiometry of receptor and coactivator. In crystal structures, each receptor was bound to a coactivator peptide. Quite probably this could be to increase the symmetry and to obtain better crystal packing (Greschik et al., 2004; Nahoum et al., 2007; Nettles et al., 2007). Based on the use of larger fragments of coactivators, such as the functional domains of Med1 and SRC1, and on mutating residues in AF2 of both receptor partners that are indispensible for coactivator binding, solution structures suggest that only 1 coactivator binds to the heterodimeric complex, more precisely to the binding partner of RXR (Rochel et al., 2011). The binding of one coactivator peptide seems to prevent the binding of a second coactivator peptide through an allosteric propagation pathway that passes through both ligand-binding pockets and the LBD dimer interface (Osz et al., 2012a).

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Hetereodimeric receptor complexes with RXR as binding partner are likely to be similar to each other since both DBD- and LBD-dimerizations are present and these determine the overall structure of the heterodimer. As a promiscuous binding partner, the structure of the RXR hinge region is more variable, while the hinges of other NRs are more structured to specifically select the specific response element for high affinity binding. For instance, because VDR–VDR and VDR–RXR dimers both bind DR3 elements, the VDR–VDR homodimer is proposed to bind like the full size VDR–RXR heterodimeric complex (Fig. 5) (Orlov et al., 2012). The structure of both hinges in the VDR–VDR homodimer remains however speculative. Perhaps one of them is more flexible and resembles the hinge of RXR as is the case in the VDR–RXR heterodimer complex or perhaps the rigidity of the hinge of VDR is forcing the VDR homodimer in a less optimal binding conforma-

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Fig. 5. Extrapolated structures for non-steroid receptors. The TR–RXR (DR4) heterodimer, the RAR–RXR (DR2) heterodimer and the VDR–VDR (DR3) homodimer based on the solution structures of several RXR heterodimers. DBDs are depicted as triangles, LBDs as circles, coactivator peptides as green cilinders. DRn are response elements composed of a direct repeat of two half-sites, spaced by n nucleotides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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tion which can explain why DR3 elements are bound with a lower affinity by VDR–VDR than by VDR–RXR. The VDR–VDR homodimer was reported to preferentially bind other types of response elements, such as DR6 elements, over DR3 elements (Carlberg et al., 1993); in this case the 6-nucleotide spacer could potentially solve the constraints due to helix rigidity. The full size TR–RXR heterodimer (Fig. 5) could also be similar to the VDR–RXR heterodimer since the location of the CTE a-helix of TR corresponds to the location of the VDR CTE a-helix (Fig. 3), , even when it binds a DR4 element (Orlov et al., 2012). Steroid receptors, on the other hand, are likely to bind to DNA in a different manner than heterodimeric nuclear receptors, because their dimerization is believed to be restricted to the second zinc finger interface and interactions between the NTD and the LBD. However, communications between the LBD and the DBD of steroid receptors have also been suggested (Helsen et al., 2012a; Huang et al., 2013). For the LBD–DBD communications between PPARc and RXRa, certain PPARc–LBD-specific features are involved. While the overall 3-dimensional conformation of the LBDs is similar for all nuclear receptors, subtle differences in secondary structure elements (helices or b-strands) can be found when aligning the crystal structure of the LBD of a steroid receptor to the LBD of a non-steroid receptor. The AR for instance lacks helix 2 while the PPARc receptor contains 2 helical segments (H2a and H2b) and a b-strand in this region (Chandra et al., 2008; Helsen et al., 2012a). The linker between helix 5 and helix 6 also presents important structural variation between non-steroid nuclear receptors

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(PPARc, TR and VDR) and steroid receptors (AR, MR, PR, GR and ER) (Garcia-Vallve and Palau, 1998). It is exactly this surface (helix 2 and the linker between helix 5 and 6) that is involved in the communication between the LBD of PPARc and the DBD of RXRa in the crystal structure of the PPARc–RXRa heterodimer on its DNA response element (Chandra et al., 2008). For the ER, computational methods have suggested that helix 12 and a region determined by helix 3, 4 and 5, all located in the LBD, could be involved in the stabilization of DNA binding via LBD–DBD cross-talk (Huang et al., 2013). The question rises thus whether the steroid receptors would display a similar LBD–DBD communication as was reported for the heterodimeric non-steroid receptors. First of all, while for the dimerization of steroid receptor LBDs (except for ER) very different formats have been proposed based on X-ray crystallography (Bledsoe et al., 2005, 2002; Zhang et al., 2005), most other data point at weak LBD-dimerizations at best. Steroid receptors bind to DNA as homodimers with their interacting DBDs in a near symmetrical head-to-head conformation. However, the two half-sites in a GRE, PRE, ARE and MRE are known to be unequal: the upstream 50 -AGAACA-30 -like hexamer is always bound with a higher affinity, while the sequence of the downstream hexamer is less constrained in so far that it sometimes appears as if the receptors are binding to a single hexamer (Dahlman-Wright et al., 1990; Shaffer et al., 2004). Point mutations in the sequence downstream of these hexamers, however, always affect receptor binding and activity (Denayer et al., 2010; Verrijdt et al., 1999). The binding to the downstream half-site is most likely stabilized via dimerization by the second zinc fingers and by allosteric signals coming from the LBD via the CTE and the DBD (Haelens et al., 2007), explaining the lower sequence stringency in the downstream part of the response elements. Interestingly, when looking for palindromic GRE-like sequences, the consensus elements for GR and AR are indeed symmetrical, but when looking for sequence motifs in unbiased ways, more asymmetrical consensus elements were described (Watson et al., 2012; Sahu et al. Cancer res 2013) (unpublished results of the group of Jänne OA and the group of Yamamoto KR). Since the VDR and the HNF-4a are non-steroid receptors that bind as homodimers on their response element (a DR3 and a DR1, respectively), a hypothetical model for steroid receptors could be derived from them (Fig. 6). An important observation for these complexes is that it is not symmetrical despite the binding of 2 identical subunits. There is a clear asymmetric position of both LBDs above the upstream DBD. It is very likely that homodimeric steroid receptors also do not form symmetrical DNA-binding complexes. For ER which also presents LBD dimerization, one could imagine a binding mode similar to the VDR-homodimer or to the HNF-4a-homodimer with a correction for spacer length (Fig. 6). For AR, on the other hand, the positions of the LBDs are less certain due to the lack of LBD dimerization and the undefined role of the NTD in the DNA binding complex (Fig. 6). In conclusion, the structures of the non-steroid nuclear receptors cannot be extrapolated easily to the steroid receptors among others because they lack the LBD dimerization and have specific DBD dimerizations. However, these first structures of full size receptors can serve as inspiration points. The differences between the two published crystal-based structures indicate that the relative positioning of LBD and DBD can vary, despite of similarities of the individual receptor domains. Obviously, the DNA sequence will be an important organizer of the receptor dimers. Moreover, the impact of the NTDs on the structures still remains elusive, although direct and indirect communications are well described (Pippal et al., 2009; Takimoto et al., 2003; Tetel et al., 1999) (Callewaert et al., 2003). Finally, the flexibility of the hinge regions should not be interpreted as merely enabling adaptation to

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Fig. 6. Hypothetical full-size binding mode of steroid receptors. The hypothetical structures are based on the open conformation of PPAR–RXR (left) and the closed HNF-4a homodimer (right). DBDs are depicted as triangles, LBDs as circles, coactivator peptides as green cilinders. IR3 is a response element composed of two inverted half-sites, spaced by 3 nucleotides. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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different dimerizations of the DBDs and LBDs, but rather as integrators and facilitators of allosteric signaling. Since the latter will be very dependent on the context (DNA sequence, posttranslational modifications and complex-binding) it is of high importance to gain more structural and functional insights in the DNA-bound receptor dimers, under these different conditions.

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