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Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors

Abstract

Members of the nuclear receptor (NR) superfamily of transcription factors modulate gene transcription in response to small lipophilic molecules1. Transcriptional activity is regulated by ligands binding to the carboxy-terminal ligand-binding domains (LBDs) of cognate NRs. A subgroup of NRs referred to as ‘orphan receptors’ lack identified ligands, however, raising issues about the function of their LBDs2. Here we report the crystal structure of the LBD of the orphan receptor Nurr1 at 2.2 Å resolution. The Nurr1 LBD adopts a canonical protein fold resembling that of agonist-bound, transcriptionally active LBDs in NRs3, but the structure has two distinctive features. First, the Nurr1 LBD contains no cavity as a result of the tight packing of side chains from several bulky hydrophobic residues in the region normally occupied by ligands. Second, Nurr1 lacks a ‘classical’ binding site for coactivators. Despite these differences, the Nurr1 LBD can be regulated in mammalian cells. Notably, transcriptional activity is correlated with the Nurr1 LBD adopting a more stable conformation. Our findings highlight a unique structural class of NRs and define a model for ligand-independent NR function.

Main

Roughly half of the mammalian NRs and most invertebrate NRs are currently classified as orphans because their bona fide endogenous ligands have not been identified. The search for ligands for orphan NRs has become the subject of intense investigation and debate. The successful identification of fatty acids, oxysterols and bile acids as naturally occurring agonists of the peroxisome proliferator-activated receptors (PPARs), the liver X receptors and the farnesoid X receptor, respectively, supports the notion that all NRs are regulated by ligands4,5. However, efforts to identify ligands for many other NRs have met with less success and structural studies of orphan receptors have suggested that some NRs such as hepatocyte nuclear factor 4α (HNF4α) and HNF4γ, although capable of binding ligands, may not be ligand-regulated6,7. But definitive evidence of a NR that is not ligand-regulated is still lacking.

Nurr1 (also known as NR4A2), an orphan NR lacking identified ligands, belongs to the nerve growth factor-induced clone B (NGFI-B) subfamily of orphan receptors that can interact with DNA as monomers or homodimers. Nurr1 and NGFI-B can also interact with DNA as heterodimers with retinoid X receptors (RXRs). Nurr1 is essential for the development of dopamine neurons in mice8,9,10 and is linked to cases of familial Parkinson's disease in humans11. Although no ligand for Nurr1 has been identified, its LBD shows constitutive and cell-type-specific activity that is dependent on an unusual sequence in the activation function-2 (AF2) region12. To facilitate identification of a putative Nurr1 ligand and to characterize further the mechanisms of Nurr1 transcriptional activation, we have determined the structure of the Nurr1 LBD by X-ray crystallography.

The overall structure of the Nurr1 LBD (Fig. 1a) adopts the characteristic canonical fold of NR LBDs3,13. Globally, Nurr1 LBD is most similar to the LBD of the holo retinoic acid receptorγ (RARγ)14 but superimposes relatively well with other ligand-bound LBDs (Fig. 1b), such as the 17β-oestradiol (E2)-bound oestrogen receptor α (ERα)15. In this conformation, the AF2 helix folds back towards the body of the LBD and packs against helices H3, H4 and H10, with its hydrophobic residues protruding into the core of the LBD. Evidently, a salt bridge and several hydrophobic interactions between helices H12 and H11 are crucial for stabilizing the AF2 helix in a conformation that would otherwise be achieved only through ligand interactions in holo NRs (Fig. 1c). These structural observations explain two previously reported mutations12, namely, that both D589A and F592A mutants abolish AF2-dependent transactivation by monomeric Nurr1 in human embryonic kidney 293 cells. Thus, the tertiary structure of the Nurr1 LBD maintains an architecture that is very similar to that of ligand-bound NR LBDs; however, the AF2 helix conformation observed in Nurr1 is stabilized by intramolecular interactions in the absence of any ligand.

Figure 1: Ribbon representations of NR LBD structures.
figure 1

a, Nurr1 LBD contains a three-layered antiparallel α-helical sandwich formed by 12 α-helices (H1–H12) and one β-sheet of two strands (S1, S2). The AF2 helix (H12) is shown in red. b, Superposition of the Nurr1 LBD in magenta, with holo RARγ (PDB code: 2LBD) in cyan and holo ERα (PDB code: 1ERE) in yellow. The view is rotated by 90° relative to a. c, Interactions between H11 and H12 that position and stabilize H12 in the transcriptionally active conformation in Nurr1 LBD. F592 from H12 forms an aromatic stacking interaction with F574 from H11, whereas K577 from H11 forms a salt-bridge with D589 from H12.

The NR ligand-binding pocket (LBP) is located in the lower part of the LBD. Ligand recognition and the specificity of ligand binding are largely determined by the size and shape of this pocket, which is altered by the side chains that constitute its lining. The LBDs of NRs reported so far have a pocket of varying size, with PPARγ having the largest cavity16 and oestrogen-receptor-related (ERR3) having the smallest17. Unexpectedly, the Nurr1 LBD structure shows that it contains no ligand-binding cavity (Fig. 2a). Instead, several tightly packed, bulky hydrophobic residues occupy the space that is available for ligand binding in other NRs. The six key hydrophobic residues that contribute to filling this space completely block the ligand passage behind helix H3 (Fig. 2b).

Figure 2: No ligand-binding cavity in Nurr1.
figure 2

a, The σA-weighted 2FoFc electron density map (contoured at 1.5 σ) of the Nurr1 LBP behind α-helix H3. b, The six residues that completely block the ligand passage behind H3 (F406 and L410 from H3, L444 and F447 from H5, F479 from loop 6–7 and W482 from H7) are shown in green. The pocket is further sealed by residues L409 from H3, L451 from H5, I483 and I486 from H7 and F464 from the β-turn on one side the structure, and residues I573 from H11, and I588 and L591 from H12 on the other side, shown in pink and yellow, respectively. The internal surface of the protein, after removing these side chains, is shown in grey.

These findings provide evidence of an NR LBD lacking a ligand-binding cavity and thus show the existence of a ligand-independent orphan NR. The NGFI-B subfamily also includes the mammalian receptors NGFI-B (NR4A1) and Nor1 (NR4A3) and the Drosophila homologue DHR38 (ref. 18). The important hydrophobic residues (Fig. 2b) that fill the space occupied by ligands in other NRs are all conserved in this subfamily. Thus, the absence of an LBP is predicted for all members of the NGFI-B subfamily, suggesting that they are all ligand-independent transcription factors.

Although the position of H12 in this ligand-free structure indicates that the protein is in a transcriptionally active conformation, a cognate surface formed by H12 and parts of H3 and H4 for cooperative interactions with coactivators is distinctly different from the surface found in other NRs. In the active conformation, a ‘charge clamp’ is normally formed between a glutamic acid residue from H12 and a lysine residue from H3 for the purpose of interacting with the amino and carboxy termini, respectively, of a helix in a coactivator polypeptide containing an LXXLL motif16,17,19. As predicted by sequence alignment, the Nurr1 LBD shows a reversed charge clamp formed by K590 from H12 and E422 from H3 (Fig. 3). K590 is unlikely to be able to perform such a role, however, because it is engaged in a salt bridge with E440 from H4.

Figure 3: The region of the coactivator-binding site.
figure 3

ad, The residues forming the coactivator interaction surface in RARγ (a) are coloured white and the equivalent residues in Nurr1 (b) are coloured yellow. The corresponding electrostatic potential surfaces are shown in c and d, respectively. Regions of positive potential are blue, negative potential is red and neutral hydrophobic areas are white. K246 and E414 form the ‘charge clamp’ residues in RARγ. e, Mutations introduced to make Nurr1 resemble more the coactivator- or corepressor-interacting surface in other NRs do not change the transcriptional activity. Mutants were tested in transfected 293 cells for their ability to activate a co-transfected luciferase reporter gene containing three copies of the Nurr1-binding sites.

More strikingly, the coactivator hydrophobic binding cleft seen in other NRs is completely transformed into a charged surface. The most substantial disruption to the coactivator-binding surface results from the side-chain packing of R418, which folds directly into the shallow groove and makes van der Waals contacts with F439 from H4 at the floor of the groove (Fig. 3b, d). Thus, the coactivator interaction surface observed in other NRs is significantly different in Nurr1. These findings indicate that Nurr1 is devoid of the capability to recruit and to interact with a coactivator or corepressor through a ‘classical’ coactivator-binding site. Indeed, the Nurr1 LBD does not interact with several tested NR coactivators including SRC-1, ACTR, PGC-1 and CBP (as assessed by two-hybrid interaction assays and reporter gene analyses; unpublished data), and coregulatory proteins that interact with the Nurr1 LBD remain to be identified.

We used mutagenesis to determine whether the coactivator-binding site in Nurr1 could be restored by simply modifying the characteristics of residues participating in the formation of the site. A complete reversal of the reversed charge clamp through the mutations K590E and E422K did not change the activity, and mutations made in this region to make Nurr1 resemble more the coactivator- or corepressor-interacting residues in other NRs did not affect activity (Fig. 3e). The structural observations, together with these mutagenesis data, indicate that there must be an alternative coactivator- or corepressor-binding surface in Nurr1. Thus, H12 most probably has a different role in Nurr1 from its role in other NRs, providing stability to the LBD rather than a direct interaction surface for coregulatory proteins.

Given that the Nurr1 LBD has no ligand-binding cavity and lacks a ‘classical’ coactivator- or corepressor-binding surface, how is Nurr1 transcriptional activity regulated? As previously observed12, both Nurr1 and a derivative in which the Nurr1 LBD was fused to the Gal4 yeast transcription factor DNA-binding domain (DBD) were considerably more active in 293 cells than in human chorion carcinoma JEG-3 cells (Supplementary Fig. 1). We used an assembly assay (Fig. 4a) based on the conditional assembly of LBD fragments20 to explore the mechanisms that underlie these differences. For ligand-bound receptors, ligands and corepressors stimulated the interaction of the two LBD fragments and this interaction was detected by the increased transcription of a reporter construct containing Gal4-binding sites. Notably, the difference in transcriptional activity of Nurr1 in the two cell lines strongly correlated with specific assembly of the H1 and H3–H12 fragments of Nurr1 LBD, indicating that this domain is stabilized in 293 cells (Fig. 4b).

Figure 4: The stability of the Nurr1 LBD correlates with cell-specific transcriptional activity.
figure 4

a, Representation of the mammalian two-hybrid LBD assembly assay. H1 of an LBD is fused to the Gal4 yeast transcription factor DBD and the remainder of the LBD (H3–H12) is fused to the strong activation domain (VP16) from herpes simplex virus. b, Cell-specific interactions between Nurr1 LBD H1 and the remainder of the Nurr1 LBD (H3–H12) are correlated with LBD-dependent transcriptional activity.

As the evidence showed that Nurr1 LBD can adopt different conformations correlating with its transcriptional activity, we investigated whether other signalling pathways might regulate this transition. As both Nurr1 and ligands for the signalling receptor tyrosine kinase Ret are important in midbrain dopamine cells, we examined the effect of a constitutively active oncogenic derivative of Ret (RetC634R) in co-transfection experiments. RetC634R had a profound negative effect on the ability of Nurr1 to activate a reporter gene in 293 cells, but not in JEG-3 cells (Fig. 5a and data not shown). Parallel experiments using a Nurr1 mutant that was unable to form dimers with RXR (Nurr1Dim)21 showed that this inhibition by RetC634R was not an indirect effect mediated through RXR (Fig. 5b).

Figure 5: Receptor tyrosine kinase activated intracellular signalling modulates Nurr1 LBD activity and stability.
figure 5

a, Constitutively active Ret (RetC634R) inhibits Nurr1 activation of an NGFI-B response element-containing luciferase reporter in a dose-dependent manner. b, c, RetC634R-induced inhibition is equally effective on wild-type Nurr1 and a dimerization-defective Nurr1 mutant (b) and affects Nurr1 AF2-dependent transcriptional activity (c). d, Coexpression of RetC634R destabilizes Nurr1 intramolecular interactions.

In addition to the AF2 domain, Nurr1 contains an activation function-1 (AF1) domain in the N-terminal region, and a deletion of the N-terminal 83 amino acids of Nurr1 abolishes the activity of AF1 (ref. 12). We found that this deletion mutant was still efficiently repressed by RetC634R, whereas a mutant containing a deletion of the AF2 domain was not, showing that the AF2 domain, but not the AF1 domain, is inhibited by RetC634R (Fig. 5c). In addition, when the Nurr1 LBD was tested in a fusion with the Gal4 DBD (Gal4–Nurr1), activation was efficiently repressed by RetC634R. As Ret signals through, for example, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase pathways, we used specific kinase inhibitors to show that inhibition of the Nurr1 AF2 domain is dependent on MAPK activation (Supplementary Fig. 2).

The influence of Ret-dependent signalling on Nurr1 LBD was assessed using the assembly assay described above. Notably, interaction between the H1 and H3–H12 fragments of Nurr1 LBD was abolished by co-transfection with RetC634R, showing that the active conformation of Nurr1 LBD is destabilized as a result of Ret signalling (Fig. 5d). Although experiments using the assembly assay provide evidence for structural transitions correlating with transcriptional activity, the mechanism remains to be elucidated. Evidence for a similar regulation of Nurr1 activity and LBD stability was obtained in experiments from neural cell lines (data not shown). Thus, our data show that AF2-dependent transcriptional activity of Nurr1 can be efficiently inhibited by cross-talk through Ret-mediated intracellular signalling and that such a regulatory influence is associated with a structural transition in the Nurr1 LBD.

In conclusion, our data converge to show that Nurr1 activity is correlated with regulated, ligand-independent stabilization of the Nurr1 LBD. The NGFI-B subfamily represents a highly conserved group of NRs22; thus, the finding that Nurr1 lacks ligand-binding capacity is in accordance with the idea that ligand binding has been independently acquired in several NRs during evolution and that ancestral NRs were not regulated by ligand binding23. The orphan receptor steroidogenic factor 1 (SF-1) may be an additional example of a NR that is regulated through ligand-independent mechanisms, as previous data have shown that the LBD activity and conformation of SF-1 can be regulated as a result of cross-talk with the MAPK signalling pathway24. In addition, homology modelling and mutagenesis studies of the Rev-ErbA subfamily of orphan NRs suggest that they might be examples of ligand-independent transcription factors lacking a cavity for ligand binding, allowing them to constitutively bind corepressor proteins25. Notably, however, our data provide unequivocal evidence of ligand-independent regulation of an orphan NR LBD.

Methods

Protein preparation

We cloned the human Nurr1 LBD (residues 328–598) by polymerase chain reaction (PCR) into a pET-15b vector (Novagen) with an N-terminal His6 tag. The protein was expressed in Escherichia coli BL21(DE3) cells (Invitrogen), which were grown in either 2YT or minimum medium supplemented with seleno-methionine (SeMet). After two steps of purification by a Ni2+ NTA-agarose column (Qiagen) and a Mono-S cation exchange column (Pharmacia), the N-terminal His tag was removed by thrombin. The protein was further purified by another Ni2+ NTA-agarose column and a Mono-Q anion exchange column (Pharmacia), and finally using a Superdex 75 gel filtration column (Pharmacia). We concentrated the purified protein to 5–7 mg ml-1 in 20 mM Tris-HCl (pH 7.9), 100 mM NaCl, 2 mM EDTA and 5 mM dithiothreitol for crystallization.

Crystallization

The Nurr1 LBD crystals were grown at 20 °C in either a hanging drop or a sitting drop configuration with 2.5 µl of the protein solution and 2.5 µl of well solution containing 18% (w/v) PEG3350, 0.1 M HEPES (pH 6.5) and 0.2 M KBr. We transferred the crystals into a well solution containing an additional 20% (w/v) of ethylene glycol and then flash-froze them in liquid nitrogen.

Data collection, structure determination and refinement

We collected X-ray diffraction data sets on both a RU-H3RHB generator/RAXIS-IV detector (Rigaku) and on a synchrotron source (beamline 5.0.2) at the Advanced Light Source (ALS) in Berkeley, California. The best diffraction resolution of 2.2 Å was obtained at ALS. All data were integrated using MOSFLM and scaled using SCALA in the CCP4 suite of programs (ref. 26). Crystals grew in two different space groups, P31 and P3121.

We resolved the Nurr1 LBD structure by two-wavelength (inflection and remote) multiple anomalous dispersion (MAD) phasing with SOLVE27 and SHARP28 using protein incorporating SeMet and the crystal form with space group P3121. Non-crystallographic symmetry (NCS) averaging using program DM in CCP4 was able to produce a traceable map of high quality. A model was built using the program O29 and refined with REFMAC5 in CCP4. A higher-resolution data set from a crystal belonging to space group P31 was used to complete the final refinement using CNX (ref. 30 and Supplementary Table 1).

The main chain and side chain atoms were visible for residues 363–390 and 399–598 in the final electron density maps for all six molecules in the asymmetric unit in space group P31. Although the first 35 amino acids of the N terminus (residues 328–362) were not visible for five molecules, one molecule did show good quality electron density for residues 334–347. This small fragment served as a centre for the packing of the six molecules.

Chemicals

We purchased PD 98059 and LY 294002 from Calbiochem and dissolved it in dimethyl sulphoxide according to the manufacturer's recommendations.

Cell culture

Human embryonic kidney 293 cells and human chorion carcinoma JEG-3 cells were maintained in Dulbecco's modified Eagle's medium and in minimum essential medium (Invitrogen), respectively. Media were supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37 °C under 5% CO2 humidified atmosphere.

Transfections and constructs

Transfections were done using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's recommendations. Typically, 0.5 µg of DNA per well was transfected into cells in 24-well plates for 3–5 h, after which the cells received fresh medium containing FCS (10% final concentration) and chemicals as indicated. Cells were cultured for an additional 20 h and then collected and assayed for luciferase and β-galactosidase activity. Each transfection was done in triplicate or quadruplicate, and each experiment was repeated at least three times. We found that expression of Nurr1 and Nurr1 mutants was equivalent in JEG-3 and 293 cells. Results show the mean ± s.e.m. of representative experiments. The constructs used in this study have been described12,21. Expression vectors pcDNA3 RetC634R and pcDNA3 RetY905F were a gift from C. Ibanez and contain the coding cDNA sequences of a constitutively active and an inactive mutant of the human c-ret, respectively.

Nurr1 LBD assembly assay

We assessed structural changes in the Nurr1 LBD using a modified version of a described LBD assembly assay20. The expression plasmid pCMX-Gal4–Nurr1-H1 contains the sequence encoding residues 350–398 (corresponding to H1) cloned in frame with the sequence encoding the DBD of the yeast Gal4 transcription factor (residues 1–147). The expression plasmid pCMX-VP16-H3–H12 contains the sequence encoding residues 399–598 (corresponding to H3–H12) cloned in frame with the sequence encoding the activation domain of the VP16 protein from herpes simplex virus.

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Acknowledgements

We thank P. Cao for MS analysis, J. Lehmann, A. Shiau, B. Shan, C. Ibanez, A. Mata and Ö. Wrange for discussions and advice. This work was supported in part by the Göran Gustafsson Foundation, The European Union Research Training Network and the Swedish Foundation for Strategic Research. The Advanced Light Source at the Lawrence Berkeley National Laboratory is supported by the Director, Office of Science, Office of Basic Sciences, Materials Sciences Division, of the US Department of Energy.

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Wang, Z., Benoit, G., Liu, J. et al. Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature 423, 555–560 (2003). https://doi.org/10.1038/nature01645

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