1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics

Molecular basis of ligand-dependent Nurr1-RXRα activation

  1. Xiaoyu Yu
  2. Jinsai Shang
  3. Douglas J Kojetin  Is a corresponding author
  1. Skaggs Graduate School of Chemical and Biological Sciences at Scripps Research, United States
  2. Department of Integrative Structural and Computational Biology, Scripps Research and UF Scripps Biomedical Research, United States
  3. Department of Molecular Medicine, Scripps Research and UF Scripps Biomedical Research, United States
https://doi.org/10.7554/eLife.85039
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  1. Xiaoyu Yu
  2. Jinsai Shang
  3. Douglas J Kojetin
(2023)
Molecular basis of ligand-dependent Nurr1-RXRα activation
eLife 12:e85039.
https://doi.org/10.7554/eLife.85039
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8 figures, 2 tables and 2 additional files

Figures

Figure 1
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Contribution of retinoid X receptor alpha (RXRα) domains on repressing Nurr1 transcription.

(a) General scheme of the cellular transcriptional reporter assay. (b) 3xNBRE-luciferase assay performed in SK-N-BE(2)-C cells; see Figure 1—source data 1 for data plotted. Data are normalized to empty vector control (n=9 replicates), shown as a box and whiskers plot with boundaries of the box representing the 25th percentile and the 75th percentile, and representative of two or more independent experiments. Statistical testing was performed and p-values were calculated using the Brown-Forsythe and Welch multiple comparisons test of the FL Nurr1 + RXRα constructs conditions relative to FL Nurr1 control condition.

Figure 1—source data 1

Nurr1+ retinoid X receptor alpha (RXRα) truncated construct luciferase reporter data.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig1-data1-v2.zip
Figure 2
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Retinoid X receptor alpha (RXRα) ligands used in this study.

Grouped by pharmacological phenotype, the set includes ligands that classically activate (agonists) or block (antagonists) activation of RXRα homodimers; a mixed activity modulator (LG100754) that antagonizes RXRα homodimers and activates PPARγ-RXRα and RAR-RXRα heterodimers; and two selective activators of Nurr1-RXRα heterodimers (BRF110 and HX600).

Figure 3
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Effect of retinoid X receptor alpha (RXRα)-binding ligands on Nurr1-RXRα transcription.

(a) General scheme of the Nurr1-RXRα/3xNBRE-luciferase cellular transcriptional reporter assay. (b) Nurr1-RXRα/3xBNRE-luciferase transcriptional reporter assay performed in SK-N-BE(2)-C cells treated with RXRα ligand (1 µM) or DMSO (dotted line); see Figure 3—source data 1 for data plotted. Data are normalized to DMSO (n=9 replicates), represent the mean ± s.d., and representative of two or more independent experiments. Statistical testing was performed and p-values were calculated using the Brown-Forsythe and Welch multiple comparisons test relative to DMSO control treated condition.

Figure 3—source data 1

Retinoid X receptor alpha (RXRα) ligand treated Nurr1-RXRα/3xNBRE3-luciferase reporter data.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig3-data1-v2.zip
Figure 4
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Compound profiling for pharmacological retinoid X receptor alpha (RXRα) agonism and correlation to Nurr1-RXRα agonism.

(a) General scheme of the RXRα ligand-binding domain (LBD) time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator peptide interaction assay. (b) TR-FRET ratio measured in the presence of DMSO (dotted line) or compound (2–4 µM); see Figure 4—source data 1 for data plotted. Data are normalized to DMSO control (n=3 biological replicates), represent the mean ± s.d., representative of two or more independent experiments. Statistical testing was performed and p-values were calculated using the Brown-Forsythe and Welch multiple comparisons test relative to the DMSO control treated condition. (c) General scheme of the RXRα/3xDR1-luciferase cellular transcriptional reporter assay. (d) RXRα/3xDR1-luciferase transcriptional reporter assay performed in HEK293T cells treated with compound (1 µM) or DMSO control (dotted line); see Figure 4—source data 2 for data plotted. Data normalized to DMSO (n=6 replicates), represent the mean ± s.d., and representative of two or more independent experiments. Statistical testing was performed and p-values were calculated using the Brown-Forsythe and Welch multiple comparisons test relative to DMSO control treated condition. (e) Correlation plot of RXRα LBD TR-FRET data vs. Nurr1-RXRα cellular transcription data. (f) Correlation plot of RXRα transcriptional reporter data vs. Nurr1-RXRα cellular transcription data. (g) Correlation plot of RXRα transcriptional reporter data vs. RXRα LBD TR-FRET data. Pearson (rp) and Spearman (rs) correlation coefficients and statistical significance testing are reported above the correlation plots.

Figure 4—source data 1

Retinoid X receptor alpha (RXRα) ligand treated RXRα ligand-binding domain (LBD) time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator interaction data.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig4-data1-v2.zip
Figure 4—source data 2

Retinoid X receptor alpha (RXRα) ligand treated RXRα/3xDR1-luciferase reporter data.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig4-data2-v2.zip
Figure 5
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Compound profiling for effects on Nurr1-retinoid X receptor alpha (RXRα) ligand-binding domain (LBD) heterodimer affinity and correlation to Nurr1-RXRα agonism.

(a) General scheme of the Nurr1-RXRα LBD isothermal titration calorimetry (ITC) experiment. (b) Nurr1-RXRα LBD heterodimer affinities (log M) in the presence of DMSO (dotted line) or compound determined from the fit of the ITC data (n=2 replicates, except n=1 for IRX4204 and PA425) and represent the mean ± s.d.; see Table 1 and Figure 5—source data 1 for data plotted. Statistical testing was performed and p-values were calculated using ordinary one-way ANOVA relative to DMSO control treated condition. (c) Correlation plot of ITC determined Nurr1-RXRα LBD heterodimer KD log M values vs. Nurr1-RXRα cellular transcription data. (d) Correlation plot of ITC determined Nurr1-RXRα LBD heterodimer KD log M values vs. fitted binding enthalpy (ΔH, which is the ΔHAB component of the homodimer competition model; see Materials and methods section for details). (e) Correlation plot of ITC determined Nurr1-RXRα LBD heterodimer KD log M values vs. calculated binding entropy (TΔS). Pearson (rp) and Spearman (rs) correlation coefficients and statistical significance testing are reported above the correlation plot.

Figure 5—source data 1

Raw isothermal titration calorimetry (ITC) thermograms and fitted data.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig5-data1-v2.zip
Figure 6 with 3 supplements
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Compound profiling for effects on Nurr1-retinoid X receptor alpha (RXRα) ligand-binding domain (LBD) conformational properties in solution and correlation to Nurr1-RXRα agonism.

(a) 2D [1H,15N]-TROSY HSQC data of 15N-labeled Nurr1 LBD heterodimerized with unlabeled RXRα LBD in the presence of RXRα ligands focused on the NMR peak of Thr411. The upper inset shows an overlay of 15N-labeled Nurr1 LBD monomer (200 µM) vs. 15N-labeled Nurr1 LBD-unlabeled RXRα LBD heterodimer (1:2 molar ratio) to demonstrate the shift of the Thr411 peak between monomer (m) and heterodimer (hd) forms; see Figure 6—figure supplement 1 for full spectral overlays. (b) Simulated 1H NMR lineshape analysis of Nurr1 LBD residue Thr411 showing the influence of ligand-induced weakening of Nurr1-RXRα LBD heterodimerization affinity; see Figure 6—source code 1 for calculation input files. (c) NMR estimated Nurr1 LBD monomer populations from the 2D NMR data (n=2 replicates, except n=1 for IRX4204 and PA425) and lines above the replicate values represent the mean ± s.d.; see Figure 6—source data 1 for data plotted. Statistical testing was performed and p-values were calculated using ordinary one-way ANOVA relative to apo/DMSO control treated condition. (d) Correlation plot of Nurr1-RXRα cellular transcription data vs. NMR estimated Nurr1 LBD monomer populations. (e) Correlation plot of ITC determined Nurr1-RXRα LBD heterodimer KD log M values vs. NMR estimated Nurr1 LBD monomer populations. Pearson (rp) and Spearman (rs) correlation coefficients and statistical significance testing are reported above the correlation plots. (f) Analytical size exclusion chromatography (SEC) analysis of Nurr1-RXRα in the presence of RXRα ligands (solid colored lines) relative to Nurr1 LBD monomer (dotted black line) and Nurr1-RXRα LBD heterodimer (solid black line); see Figure 6—figure supplement 2 for all SEC data organized by ligand.

Figure 6—source code 1

Input files for NMR LineShapeKin simulated NMR data analysis in MATLAB (two input files and one readme file).

https://cdn.elifesciences.org/articles/85039/elife-85039-fig6-code1-v2.zip
Figure 6—source data 1

Retinoid X receptor alpha (RXRα) ligand treated Nurr1-RXRα ligand-binding domain (LBD) NMR-observed monomer species.

https://cdn.elifesciences.org/articles/85039/elife-85039-fig6-data1-v2.zip
Figure 6—figure supplement 1
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Full overlay of 2D [1H,15N]-TROSY-HSQC data of 15N-labeled Nurr1 ligand-binding domain (LBD) (200 μM) in monomeric (m) and heterodimer (hd) forms with retinoid X receptor alpha (RXRα) LBD (400 μM).
Figure 6—figure supplement 2
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Analytical size exclusion chromatography (SEC) profiles of Nurr1 ligand-binding domain (LBD) (monomer), retinoid X receptor alpha (RXRα) LBD (homodimer and homotetramer), and Nurr1-RXRα LBD (heterodimer) with RXRα ligands present in the RXRα LBD-containing conditions (homodimer or Nurr1-RXRα heterodimer).
Figure 6—figure supplement 3
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SDS-PAGE analysis of fractions collected from size exclusion chromatography (SEC) analysis of Nurr1-retinoid X receptor alpha (RXRα) ligand-binding domain (LBD) heterodimer in the presence of BRF110.

Fractions corresponding to the indicated regions of the SEC chromatography are annotated, as well as the locations of the Nurr1 LBD and RXRα LBD. The bands annotated with ** correspond to a degraded RXRα LBD population that formed when these SEC samples were stored at 4°C for an extended period of time prior to SDS-PAGE analysis. See Figure 6—figure supplement 3—source data 1for raw unedited gel with and without annotation.

Figure 6—figure supplement 3—source data 1

Full raw unedited gel, without and with annotation (two JPG files).

https://cdn.elifesciences.org/articles/85039/elife-85039-fig6-figsupp3-data1-v2.zip
Figure 7
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Principal component analysis (PCA) of experimental data reveals Nurr1-retinoid X receptor alpha (RXRα) agonism and classical RXRα agonism are uncorrelated.

(a) 2D biplot containing the loadings and PC scores of the first two PCs. PC scores of the ligands are colored according to pharmacological activity (see legend) whereas the loadings (experimental variables) are colored light blue. (b) A proportion of variance plot revealing the amount of variance described by each PC individually and cumulatively.

Figure 8
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Data-informed model for activation of Nurr1-retinoid X receptor alpha (RXRα) transcription by RXRα ligands.

Tables

Table 1
Fitted and calculated isothermal titration calorimetry (ITC) Nurr1-RXRα binding affinity and thermodynamic parameters.
Ligandlog KD (M)ΔH (kcal/mol)TΔS (kcal/mol)
Averages.d.nReplicatesAverages.d.nReplicatesAverages.d.nReplicates
DMSO–5.3350.1182–5.418–5.2522.9000.24023.0702.73010.1790.401210.4629.895
BRF110–4.3760.0202–4.390–4.3625.3650.36125.1105.62011.3350.334211.09911.571
HX600–4.6620.0282–4.681–4.6427.6954.32024.64010.75014.0544.283211.02617.083
9cRA–5.0260.0602–4.984–5.0685.1100.96225.7904.43013.5393.105212.58911.344
Bexarotene–4.6190.0442–4.588–4.6505.5551.33624.6106.50011.8571.396210.87012.844
LG100268–5.0440.1062–5.119–4.9695.1350.14825.2405.03012.0170.294212.22411.809
CD3254–4.8600.0992–4.930–4.7904.8650.38925.1404.59011.4950.524211.86611.125
SR11237–5.0530.0472–5.087–5.0204.9400.96225.6204.26011.8341.026212.56011.109
UVI3003–4.5650.0212–4.580–4.5506.1252.63827.9904.26012.3532.666214.23810.467
LG100754–5.2220.0262–5.204–5.2404.2700.22624.1104.43011.3940.261211.20911.579
IRX4204–4.6730.0001–4.6738.4600.00018.46014.8350.000114.835
Rhein–5.5540.0772–5.499–5.6092.7800.39623.0602.50010.3570.290210.56310.152
HX531–6.1320.1332–6.226–6.0382.2050.02122.1902.22010.5710.160210.68410.457
Danthron–5.5240.0432–5.494–5.5542.9200.17023.0402.80010.4560.111210.53510.378
PA425–5.4170.0001–5.4174.1200.00014.12011.5100.000111.510
Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)BL21(DE3)Sigma-AldrichCMC0014Electrocompetent cells
Cell line (Homo sapiens)Human embryonic kidney epithelialATCCCRL-11268
Cell line (Homo sapiens)SK-N-BE(2) neuroblastomaATCCCRL-2271
Chemical compound, drugBRF110Sigma-AldrichCAS 2095489-35-1
Chemical compound, drugHX600Axon MedchemCAS 172705- 89-4
Chemical compound, drug9-cis-Retinoic acidCayman ChemicalsCAS 5300-03-8
Chemical compound, drugBexaroteneCayman ChemicalsCAS 153559- 49-0
Chemical compound, drugLG100268Cayman ChemicalsCAS 153559-76-3
Chemical compound, drugCD3254Cayman ChemicalsCAS 196961-43-0
Chemical compound, drugSR11237Tocris BioscienceCAS 146670-40-8
Chemical compound, drugUVI3003Cayman ChemicalsCAS 847239-17-2
Chemical compound, drugLG100754Cayman ChemicalsCAS 180713-37-5
Chemical compound, drugIRX4204MedChemExpressCAS 220619-73-8
Chemical compound, drugRheinSigma-AldrichCAS 478-43- 3
Chemical compound, drugHX531Cayman ChemicalsCAS 188844-34-0
Chemical compound, drugDanthronSigma-AldrichCAS 117-10-2
Chemical compound, drugPA425Tocris BioscienceCAS 457657-34-0
Peptide, recombinant proteinFITC-PGC1αLifeTeinAmino acid sequence: EAEEPSLLKKLLLAPANTQ, with an N-terminal FITC label and an amidated C-terminus.
Recombinant DNA reagentNurr1-ligand binding domain (LBD) (plasmid)de Vera et al., 2016Bacteria expression plasmid
Recombinant DNA reagentRXRα-ligand binding domain (LBD) (plasmid)Kojetin et al., 2015Bacteria expression plasmid
Recombinant DNA reagentpET45b(+)Novagen71327-3
Transfected construct (Photinus pyralis)3xNBRE-luciferase plasmidde Vera et al., 2016Sanger sequenced
Transfected construct (Photinus pyralis)3xDR1-luciferase plasmidHughes et al., 2014Mammalian expression plasmid, Sanger sequencedThe 3xPPRE-luciferase reporter plasmid in the referenced paper was used in our study.
Transfected construct (human)Full-length human Nurr1de Vera et al., 2016Mammalian expression plasmid, Sanger sequenced
Transfected construct (human)Full-length human RXRα in pCMV-Sport6 vectorZhang et al., 2011cMammalian expression plasmid, Sanger sequencedWe obtained this plasmid from Griffin lab at UF Scripps Institute (see referenced paper).
Recombinant DNA reagentpcDNA3.1 empty vectorThermo Fisher ScientificV790-20
Sequence-based reagentRXRα-ΔLBD-FThis paperPCR primer ordered from SigmaCAGCAGCGCCTAAGAGGACATG
Sequence-based reagentRXRα-ΔLBD-RThis paperPCR primer ordered from SigmaCATGTCCTCTTAGGCGCTGCTG
Sequence-based reagentΔNTD-RXRα-FThis paperPCR primer ordered from SigmaCCACCCCTCGAGAAACATGG
Sequence-based reagentΔNTD-RXRα-RThis paperPCR primer ordered from SigmaCCATGTTTCTCGAGGGGTGG
Gene (human)Nurr1 (NR4A2)UniprotFull length: residues 1–598; LBD: residues 353–598
Gene (human)RXRα (NR2B1)UniprotFull length: residues 1–462; LBD: 223–462
Sequence-based reagentRestriction enzymes, ligase for cloningNEBXhoI, HindIII, T4 ligase
Commercial assay or kitGibson assemblyNBEE2611L
Commercial assay or kitBritelite plus Reporter Gene Assay SystemPerkin Elmer6066769
Sequence-based reagentRXRα-LBDThis papergBlock for Gibson assemblyCCAGCACAGTGGCGGCCGCATGAAGCGGGAAGCCGTGCAGGAGGAGCGGCAGCGTGGCAAGGACCGGAACGAGAATGAGGTGGAGTCGACCAGCAGCGCCAACGAGGACATGCCGGTGGAGAGGATCCTGGAGGCTGAGCTGGCCGTGGAGCCCAAGACCGAGACCTACGTGGAGGCAAACATGGGGCTGAACCCCAGCTCGCCGAACGACCCTGTCACCAACATTTGCCAAGCAGCCGACAAACAGCTTTTCACCCTGGTGGAGTGGGCCAAGCGGATCCCACACTTCTCAGAGCTGCCCCTGGACGACCAGGTCATCCTGCTGCGGGCAGGCTGGAATGAGCTGCTCATCGCCTCCTTCTCCCACCGCTCCATCGCCGTGAAGGACGGGATCCTCCTGGCCACCGGGCTGCACGTCCACCGGAACAGCGCCCACAGCGCAGGGGTGGGCGCCATCTTTGACAGGGTGCTGACGGAGCTTGTGTCCAAGATGCGGGACATGCAGATGGACAAGACGGAGCTGGGCTGCCTGCGCGCCATCGTCCTCTTTAACCCTGACTCCAAGGGGCTCTCGAACCCGGCCGAGGTGGAGGCGCTGAGGGAGAAGGTCTATGCGTCCTTGGAGGCCTACTGCAAGCACAAGTACCCAGAGCAGCCGGGAAGGTTCGCTAAGCTCTTGCTCCGCCTGCCGGCTCTGCGCTCCATCGGGCTCAAATGCCTGGAACATCTCTTCTTCTTCAAGCTCATCGGGGACACACCCATTGACACCTTCCTTATGGAGATGCTGGAGGCGCCGCACCAAATGACTTGATCGAGTCTAGAGGGCCCG
Commercial assay or kitLanthaScreen Elite Tb-anti-His antibodyThermo Fisher#PV5895
Software, algorithmNITPIC softwareKeller et al., 2012Baseline calculation, curve integration
Software, algorithmSEDPHATBrautigam et al., 2016Estimation of binding affinity and thermodynamic parameter measurements
Software, algorithmGUSSIBrautigam, 2015Plot ITC figures
OtherNMR chemical shift assignment of Nurr1 LBDMichiels et al., 2010; de Vera et al., 2019; Munoz-Tello et al., 2020BMRB16541Published NMR peak assignment from Biological Magnetic Resonance Data Bank
Software, algorithmNMRFxNorris et al., 2016NMR data process and analysis
Software, algorithmNMR LineShapeKin version 4Kovrigin, 2012NMR lineshape analysis
Software, algorithmMATLAB R2022a via NMRboxMaciejewski et al., 2017NMR lineshape analysis
Software, algorithmPearson and Spearman correlation analysisGraphPad PrismCorrelation analysis
Software, algorithmPrincipal component analysis (PCA);GraphPad PrismCorrelation analysis
Software, algorithmANOVA multiple comparison testGraphPad PrismStatistical testing

Additional files

Supplementary file 1

gBlock sequence used to clone the retinoid X receptor alpha (RXRα) ligand-binding domain (LBD) only construct.

https://cdn.elifesciences.org/articles/85039/elife-85039-supp1-v2.docx
MDAR checklist
https://cdn.elifesciences.org/articles/85039/elife-85039-mdarchecklist1-v2.pdf

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  1. Xiaoyu Yu
  2. Jinsai Shang
  3. Douglas J Kojetin
(2023)
Molecular basis of ligand-dependent Nurr1-RXRα activation
eLife 12:e85039.
https://doi.org/10.7554/eLife.85039