Crystal structure of the NR2E3 LBD in an autorepressed conformation

NR LBDs are characterized by a canonical fold, typically comprising 12 α-helices, of which the N-terminal helices are much less conserved and defined. To determine the domain boundaries of the NR2E3 LBD, we designed four LBD expression constructs with variations at the N-terminus, starting at either G159, G170, A180, or D192 of full length NR2E3 (Figure 1). When expressed as His₆ Glutathione S-Transferase (GST)- or His ₆Sumo-fusion proteins, all of them were poorly soluble and unstable. Expression with a non-cleavable N-terminal Maltose Binding Protein (MBP) tag and C-terminal His₆ tag yielded soluble proteins that gave crystals, but these crystals did not diffract X-rays. Based on the poor behavior of these proteins and the weak conservation of the NR2E3 LBD N-terminus, we predicted that the NR2E3 LBD might not have an N-terminal helix 1, which is a characteristic of the related orphan nuclear receptor Testicular Receptor 4 (TR4). The N-terminal region of TR4 lacks a helical structure and instead adopts a long flexible loop that extends to the canonical helix 3 (Figure 1) [39].

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Figure 1. Sequence alignment of NR2E3 with other orphan receptors (COUP-TFII, TR4 and TLX) in the NR2 subgroup family and predictions on their secondary structure.

Constructs were designed with various N-terminal start points (G159, G170, A180, D192 and L217). Predicted α-helices are indicated as red cylinders below the sequences. Asterisks indicate the two charge clamp residues that are important for the correct positioning of coactivator LXXLL motifs in the cofactor binding groove.

https://doi.org/10.1371/journal.pone.0074359.g001

We thus designed an expression construct of NR2E3(217-410), which lacks the N-terminal region that corresponds to the flexible loop in TR4, as a fusion protein with a non-cleavable N-terminal MBP tag and a C-terminal non-cleavable His₆ tag. This fusion protein was expressed and purified to homogeneity in the absence of ligand (Figure 2A). The MBP tag when fused to a target protein can stabilize the target protein and greatly facilitate crystal formation of the fusion proteins [40-43]. As it is in our case, we readily crystallized the MBP-NR2E3 fusion protein in the P2 ₁2 ₁2 space group and solved its structure at a resolution of 2.8 Å by using the MBP and TR4 structures as the molecular replacement models (Figure 1B). The statistics of data collection and structure refinement are shown in Table 1.

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Figure 2. Crystal structure of the NR2E3 LBD is in an autorepressed conformation.

A. Purification of the MBP-NR2E3 LBD-His₆ fusion protein. The molecular weight of full length MBP–NR2E3LBD-His₆ is 63.6 kDa. B. Overview of the NR2E3 dimer. Each monomer is colored purple, with helix 10 (H10) colored cyan and activation function domain 2 (AF2) colored yellow. C. Front and side views of the NR2E3 LBD monomer. The secondary structure assignment is labeled according to nuclear receptor testicular receptor 4 (TR4). D. The ligand binding pocket space within the bottom half of the NR2E3 LBD is occupied by large hydrophobic side chains (shown in red stick presentation). E. Hydrophobic interactions of the NR2E3 AF2 helix within the cofactor binding site. Positively charged surfaces are shown in blue, negatively charged surfaces in red, and the uncharged, hydrophobic groove in white. F. Overlay of the NR2E3 LBD structure with the SRC1 LXXLL motif (in green) from the RXR structure (1K74). G. Overlay of the NR2E3 LBD structure with the SMRT LXXIIXXXL corepressor motif (in magenta) from the antagonist-bound PPARα structure (1KKQ).

https://doi.org/10.1371/journal.pone.0074359.g002

The crystal structure reveals a dimeric arrangement of the NR2E3 LBD (Figure 1B). Each monomer consists of a canonical antiparallel three-layer α-helical sandwich fold comprising 8 alpha helices (H3-5, H7-H10, and AF2), with a disordered region between helix 5 and helix 7 (Figures 1, 2B, and 2C). The dimer interface is a parallel coiled-coil formed by the helices 10 of both monomers (Figure 2B). Similar to the structures of the ligand-free orphan NRs TR4, Rev-erb, NUclear Receptor Related-1 protein (NURR1), Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII), Dosage-sensitive sex reversal, and Adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) [39,44-47], the apo NR2E3 LBD lacks a ligand-binding pocket. Instead, the C-terminal part of helix 10 bends and collapses into the space that corresponds to the ligand binding pocket in other NR. This space is filled with hydrophobic and aromatic residues (W257, F261, I381, I386, F390 and F391) whose bulky side chains protrude into the cavity (Figure 2D) and, in this conformation, do not provide enough space to allow ligand binding.

The kink in helix 10 and the subsequent collapse of the binding pocket of NR2E3 allows the AF2 helix, which follows helix 10, to bind in the cofactor binding site of the LBD. Hydrophobic interactions stabilize the AF2 binding in this position, with residues M400, L403, L404 and M407 in the AF2 helix forming Van-der-Waals interactions with residues from helix 3 and 4 (Figure 2E). As a result of the occupation of the cofactor-binding site, the AF2 helix would block the binding of either a coactivator or a co-repressor to the LBD and would therefore be expected to inhibit the receptor’s activity (Figure 2F and 2G). Thus, the collapsed ligand-binding pocket and the blocked coactivator binding groove indicate that the apo NR2E3 LBD adopts an autorepressed conformation.

AF2 is important for NR2E3 repression

Similar autorepressed features have been observed in the structures of the ligand-free orphan nuclear receptors Rev-erbβ (2V0V), COUP-TFII (3CJW), and TR4 (3P0U) [39,45,47] (Figure 3A). Superposition of the NR2E3 LBD to that of the above orphan nuclear receptors shows a clear similarity with root mean square deviations (rmsd) of 1.88 Å (131 C^α atoms), 1.42 Å (147 C^α atoms) and 1.38 Å (153 C^α atoms) respectively. There are noticeable differences between Rev-erbβ and NR2E3 in the position of helix 7 and in the very C-terminus, as Rev-erbβ lacks the activation function 2 helix (Figure 3A). In the absence of an AF2 motif, helix 11 provides a structural platform for the binding of the co-repressor NCoR1, which is crucial for the constitutive repressive activity of Rev-erbβ [47]. COUP-TFII and TR4 LBDs are overall very similar to that of NR2E3, but differ in their AF2 position (Figure 3A). We have previously shown that the AF2 helix is important for the transcriptional activation function of both COUP-TFII and TR4 [39,45]. Since binding of canonical NR corepressors and coactivators occurs in the hydrophobic groove formed by helices 3, 4, and AF2 [48], we were interested to test whether mutations and deletion of this region can affect NR2E3 transcriptional repression.

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Figure 3. Activation function domain 2 is important for NR2E3 repression.

A. Superposition of the alpha-C helix traces of the NR2E3 monomer (blue) with those of Rev-erbβ (red), Coup-TFII (green), and TR4 (orange). B. Schematic presentation of the domain structure of NR2E3 and of the transfection constructs. C. Gal4DBD–NR2E3LBD is a transcriptional repressor. AD293 cells were transiently transfected with either Gal4DBD (100 ng) or Gal4DBD–NR2E3LBD (10, 30, 100 ng) expression plasmids. Transcriptional activity was measured as luciferase activity. Luciferase activity was derived by normalizing firefly luciferase values to Renilla luciferase values, which was used as internal transfection control. D. The corepressor hydrophobic binding groove is formed by helix 3 (H3), helix 4 (H4), and activation function 2 (AF2). E. Effects of AF2 deletion and V232A and L253A mutations on NR2E3 repressor activity (top). Below: Relative Gal4DBD–NR2E3LBD expression levels determined by anti-Gal4DBD immunoblot. The molecular weight of full length Gal4DBD–NR2E3LBD is 42 kDa.

https://doi.org/10.1371/journal.pone.0074359.g003

In order to correlate the observed structural features with NR2E3 function, we expressed the NR2E3 LBD (residues 192-410, Figure 3B) as a chimeric protein fused to the DNA binding domain (DBD) of the yeast transcription activator Gal4. This construct was transiently transfected into AD293 cells and its effect was tested on a Gal4-responsive reporter plasmid using a luciferase assay (Figure 3B). In agreement with previous studies, this construct functioned as a transcriptional repressor [2] [37] [49] and reduced basal luciferase reporter activity in a dose dependent manner (4-5 fold reduction with 100 ng of NR2E3 expression vector; Figure 3C).

To test the role of the AF2 helix for NR2E3 activity, we expressed a C-terminally truncated LBD (residues 192-400) that lacks the entire AF2 helix. Surprisingly, the AF2-truncated LBD is expressed at the same level as the full length LBD, yet has strongly reduced repression activity (Figure 3E), suggesting that the AF2 helix is required for NR2E3 transcriptional repressor activity. To pinpoint the amino acids involved in this interaction, we created a series of site-directed mutations to substitute hydrophobic residues (I220, L228, L229, V232, L235) along helix 3 and 4 against alanine and tested these constructs in cell-based activation assays. Our data suggest that amino acids V232 on helix 3, L253 on helix 4, and the AF2 helix are important for transcriptional repression by this receptor. Since the AF2 helix blocks access to the canonical cofactor-binding site, these results further suggest the intriguing possibility that the AF2 helix in the apo conformation sterically clashes with coactivator binding, while NR2E3-specific corepressors may still be able to associate with NR2E3 via additional cofactor-binding surfaces on the receptor (see Discussion).

NR2E3 transcriptional repression activity requires the formation of a functional dimer

The formation of dimeric LBDs is one of the hallmarks observed in many NRs. Importantly, the NR2E3 LBD also forms dimers in solution as shown by size exclusion chromatography (Figure 4C). In order to determine the role of the NR2E3 dimer configuration in NR2E3 repressor function, we made single or combined mutations to replace two key residues of the coiled coil dimer interface with arginine (L372 and L375; Figure 4A and 4B). These residues are conserved in the related NRs COUP-TFII, TR4, and TLX (Figure 1) and predicted to compromise or abolish LBD dimer formation. To test this prediction, we cloned wildtype and mutant PNR LBD (amino acids 192-410) into pBind-Gal4 and pVP16 vectors for mammalian two hybrid assays. As shown in Figure 4D, the mutant LBDs failed to produce specific reporter gene activity, suggesting that both the L372 and the L375 residue are important for dimer formation. We then tested whether NR2E3 repressor activity is affected when NR2E3 dimer formation is disrupted. As shown in Figure 4E, single mutations of L372 and L375 to arginine reduced NR2E3 repression ability by >50%, and the double mutant L372R/L375R retained only 20-30% of the activity of the wild-type receptor. These results suggest that transcriptional repression by NR2E3 requires the formation of a functional LBD dimer, consistent with other studies that showed the functional form of full length NR2E3 is a dimer [2] [49], and indicate that L372 and L375 are crucial residues for forming the hydrophobic dimerization interactions.

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Figure 4. NR2E3 transcriptional repression activity requires the formation of a functional dimer.

A. Top view of the NR2E3 LBD dimer, showing the close interaction of L372 and L375 (red and orange stick models, resp.) from the helices 10 (cyan) in the dimer interface. B. A close-up view of the helices 10 in the dimer interface. C. Size Exclusion Chromatography for Bio-rad Protein Standard (left) and MBP-NR2E3 LBD (right). D. Mutation of helix 10 coiled coil interface residues abolished LBD dimerization in a mammalian two hybrid assay. Reporter gene activation by Gal4DBD–NR2E3LBD and VP16AD–NR2E3LBD wildtype and mutant expression plasmids is shown as bar graph. Cells cotransfected with pBIND-Id and pACT-MyoD were used as positive controls. E. Effects of the mutations L372R, L375R, and the double mutation L372R/L375R on NR2E3 repression activity (top). Below: Expression levels of wildtype and mutant Gal4DBD–NR2E3LBD determined by immunoblotting.

https://doi.org/10.1371/journal.pone.0074359.g004

Computer-modeling predicts a ligand pocket in the active conformation of the NR2E3 LBD

One of the goals of determining the NR2E3 structure was to determine whether it has a ligand-binding pocket and if its activity is regulated by a hypothetical ligand. As mentioned above, the ligand binding pocket of the structure we have solved is packed with hydrophobic residues and space in the pocket is further limited by the kink at the C-terminal end of helix 10. The structure of the related nuclear receptor Retinoid X Receptor (RXR/NR2B1) LBD has been solved both in the apo state [50] as well bound to the RXR agonist 9-cis retinoic acid [51]. To illustrate possible conformational changes toward a hypothetical active NR2E3 LBD structure, we overlaid the NR2E3 LBD structure with both the apo- (Figure 5A) [1LBD] and agonist-bound (Figure 5B) [1FBY] RXR LBD structures. The core of the apo-NR2E3 structure is largely similar to that of the apo-RXRα structure, but differs substantially from the structure of the RXR LBD bound to its agonist 9-cis retinoic acid, in which helix 10 is fully extended (Figure 5B). Notably, 13-cis retinoic acid, an isomer of 9-cis retinoic acid, exhibits at high concentrations limited NR2E3 agonist properties [37].

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Figure 5. Computer model of the active conformation of the NR2E3 LBD reveals a putative open ligand-binding pocket.

A. Overlay of the apo LBDs of NR2E3 (purple) and RXR (green). B. Overlay of the LBDs of apo NR2E3 (purple) and agonist-bound RXR (lime green). The main difference between the ligand binding pockets lies in the extension of helix 10 in agonist-bound RXR. C. Computer model (SWISS Model) of the NR2E3 LBD in an active conformation based on the agonist-bound RXRα structure, with helix 10 extended. A ligand binding pocket of 578 ų was found in this conformation. The pocket volume and the surface (grey mash) were calculated using the program VOIDOO. D. Close-up of the potential ligand binding pocket in the active model of the NR2E3 LBD and its surrounding residues. E. 9-cis retinoic acid (ball model), which has limited NR2E3 agonistic properties [37], fits well into the modeled ligand binding pocket.

https://doi.org/10.1371/journal.pone.0074359.g005

To probe whether the NR2E3 LBD can accommodate a hypothetical ligand, we modeled an active conformation of the NR2E3 LBD based on the structure of the agonist bound-RXRα LBD (PDB code: 1FBY), which has 39% sequence identity with the NR2E3 LBD [2]. The model of activated NR2E3 contains a ligand-binding pocket that is surrounded by the hydrophobic residues H221, F261, W257, M298, V302 and L389. The pocket has a calculated size of 578 ų (Figure 5C and 5D), a space adequate to accommodate small compounds. While 13-cis retinoic acid can be nicely fitted in the active conformation pocket (Figure 5D), the space is not able to accommodate compound 11A, a synthetic NR2E3 activator [37]. This result is consistent with a recent proposal that compound 11A does not bind directly to the NR2E3 pocket [52].

Disease-causing mutations in the NR2E3 LBD predominantly affect receptor structural integrity

Single amino acid mutations in NR2E3 have been associated with various eye diseases, but the basis by which they affect NR2E3 function is unknown. To gain insight into the function of amino acid residues altered in patients, we mapped residues deposited in the Leiden Open Variation Eye Disease Database (www.LOVD.nl/eye) on the LBD crystal structure (Figure 6A and 6B) and determined the effect of these mutations on NR2E3 repressor activity in a cell-based assay (Figure 6C). As described previously [53] [54], and confirmed here in another cell line, mutants L263P, R311Q, R336P, L353V, and M407K strongly reduced the transcriptional repressor activity of the NR2E3 LBD, consistent with these mutants being disease-causing. Patient mutations mapped mostly to helices 5, 7 and 9 (Figure 6A and 6B). Surprisingly, altered amino acids were not located in regions involved in receptor dimerization, ligand binding, or the canonical corepressor binding site at the AF2 site, regions that we would expect to affect NR2E3 transcriptional repressor activity. In order to test if the lack of repressor activity was a result of poor protein stability, we analyzed protein expression levels by immunoblotting. Expression of the NR2E3 mutants L263P, R336P, and L353V was almost abolished and expression of the remaining mutants R309G, R311Q, R334G and M407K was strongly reduced, suggesting that these mutations mainly affect NR2E3 function by reducing protein stability.

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Figure 6. Structural analysis of disease-causing mutations in NR2E3.

A. List of reported NR2E3 mutations found in patients with various eye diseases. ESCS: enhanced S-cone sensitivity syndrome, CPRD: clumped pigmentary retinal degeneration, ARRP: autosomal recessive retinitis pigmentosa. B. NR2E3 LBD mutations found in patients mapped on the receptor. The view is presented similar to the orientation shown in Figure 2C. C. Effects of eye disease mutations on NR2E3 repressor activity. Left panel: Reporter gene expression of Gal4DBD–NR2E3LBD wildtype and mutant proteins. Right panel: Expression levels of wildtype and mutant Gal4DBD–NR2E3LBD determined by immunoblotting.

https://doi.org/10.1371/journal.pone.0074359.g006

Protein preparation

The human NR2E3 LBD (NR2E3 residues 217-410) with C275S mutation was expressed as a MBP–NAAAEF linker–NR2E3(217-410)-His₆ fusion protein from the expression vector pETDuet1 (Novagen). Six liters of BL21 (DE3) cells were grown to an OD₆₀₀ of 1, followed by an induction with 100 µM of isopropyl-beta-D-thio-galactopyranoside (IPTG) at 16 °C. After overnight incubation, cells were harvested and resuspended in 100 ml of extract buffer (200 mM NaCl, 25 mM Tris, pH 8, 10% glycerol, 2 mM β-mercaptoethanol, and 200 µl of saturated PMSF). Cells were lysed using a French Press with pressure set at 1,000 Pa. Lysates were centrifuged at 13,000 rpm for 50 min and the supernatant was loaded onto a pre-equilibrated 5 ml Ni-chelating Hi-Trap column (Amersham Biosciences). The column was washed with 100 ml buffer A (200 mM NaCl, 25 mM Tris pH 8, 25 mM imidazole and 10% glycerol) and the fusion protein eluted with buffer B (200 mM NaCl, 25 mM Tris pH 8, 500 mM imidazole, and 10% glycerol). The eluted proteins was further purified by Superdex 200 gel filtration chromatography (Amersham Biosciences) in 200 mM ammonium acetate, 25 mM Tris, pH 8, 1 mM EDTA, and 1 mM dithiotreitol and concentrated to 5 mg/ml for crystallization.

Crystallization and data collection

Crystals of the MBP-NR2E3 LBD-His₆ fusion protein were grown at 20 °C in hanging drops containing 1 µl of the above protein solution and 2 µl of well buffer containing 7% polyethylene glycol 6000 (Sigma), 8% isopropanol, 10 mM Tris(2-carboxyethyl) phosphine (TCEP) and 0.1 M sodium citrate, pH 5.6. Crystals appeared within 3 days and grew to a final size of 100-300 µm on day 7. Crystals were mounted and soaked in the mother liquor solution supplemented with 20% 2,3 butanediol for cryo-protection. All crystals were flash frozen in liquid nitrogen prior to data collection.

Structure determination and refinement

The NR2E3 crystals formed in the P2 ₁2 ₁2 space group (Table 1). The datasets were collected with a MAR225 CCD detector at the ID line of sector 21 of the Advanced Photon Source at Argonne National Laboratory (Argonne, IL). The observed reflections were reduced, merged, and scaled with DENZO and SCALEPACK in the HKL2000 package [66].

The CCP4 program PHASER was used for molecular replacement [67], with the TR4 LBD (PBD code: 3P0U) [39] as a search model. The initial model was manually built and refined with CNS and the CCP4 program REFMAC [68]. All figures were prepared using PyMOL [69]. The pocket volume and surface area were calculated using VOIDOO [70].

During structure determination, molecular replacement solutions obtained from using MBP as an ensemble to search for 2 copies of MBP failed. We were only able to produce an interpretable electron density map for one MBP. Islands of electron density could be seen in the supposed area of the other MBP, but the density was not sufficient for us to solve the structure of a second MBP. However, the lack of clear density for the second MBP was not due protein truncation/proteolysis during crystallization as washed crystals loaded on a denaturing SDS-PAGE clearly showed the intact fusion protein (data not shown).

Mammalian two-hybrid assay and luciferase reporter assays

The expression plasmids were constructed by inserting NR2E3-LBD (residues 192-410) into the pBind-GAL4 vector and pACT-VP16 vectors (Promega). Transfections were performed in AD293 cells, which were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cells were plated in 24-well tissue culture plates. After overnight incubation, cells were transfected with 100 ng of pBind-GAL4-NR2E3 LBD, 100 ng of pGLuc-5, and 0.1 ng of pHRL-TK-RLuc in Opti-MEM using Extreme9 according to the manufacturer’s protocol (Promega). One day after transfection, medium was changed to fresh medium. Two days after transfection, lysates were collected and luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase as an internal transfection control. Luciferase activities were measured using an EnVision Multilabel Reader (PerkinElmer). All assays were performed in triplicate and presented with standard deviations.

Extraction of protein samples and Western blotting

Protein extracts were prepared using the CelLytic MT Cell Lysis Reagent (Sigma) and protein concentrations were determined by Bradford Assay. Proteins from lysates were separated by 4-20% gradient SDS-PAGE, transferred to nitrocellulose membrane, and detected by immunoblotting using anti-Gal4 DBD antibodies (Santa Cruz; sc-510).

Site Directed Mutagenesis

Point mutations were introduced in pGAL4-NR2E3-LBD and pACT-NR2E3-LBD constructs using the QuikChange method (Agilent). All mutant constructs were confirmed by DNA sequencing.

Statistical Analysis

Statistical analysis was performed using Excel. Comparisons were performed using Student’s independent-sample t-test. The statistical significance level was set to be p < 0.05 or p < 0.01.