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.
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).
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.
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.
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 Å3 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.
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.