Comprehensive Set of Tertiary Complex Structures and Palmitic Acid Binding Provide Molecular Insights into Ligand Design for RXR Isoforms
Abstract
:1. Introduction
2. Results
2.1. Unprecedented Identification of Palmitic Acid Binding to RXRα
2.2. Palmitic Acid Also Interacted with Other RXR Isoforms
2.3. Binding of PA in RXRs
2.4. Structural Analysis of Amino Acid Differences among RXR Isoforms
2.5. PA Acts as a Modulator of RXRs
3. Discussion
4. Materials and Methods
4.1. Expression and Purification of RXR Isoform LBDs
4.2. Crysallization and Structure Determination
4.3. Hybrid Reporter Gene Assays for RXRα, RXRβ and RXRγ
4.4. SRC-1 Recruitment Assay (HTRF)
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AD | Alzheimer’s disease |
DTT | dithiotreitol |
FI | fluorescence intensity |
FRET | fluorescence resonance energy transfer |
GRIP-1 | glucocorticoid receptor-interacting protein-1 |
HTRF | homogenous time-resolved fluorescence resonance energy transfer |
LBD | Ligand-binding domain |
MA | myristic acid |
Nurr1 | nuclear receptor related 1 |
PA | palmitic acid |
PPAR | peroxisome proliferator-activated receptor (NR1C1-3) |
RMSD | root-mean-square deviation of atomic positions |
RXR | retinoid X receptor (RXRα: NR2B1; RXRβ: NR2B2; RXRγ: NR2B3) |
SA | stearic acid |
SRC-1 | steroid receptor co-activator 1 |
THR | thyroid hormone receptor (NR1A1-2) |
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Complex a | RXRα-PA-GRIP-1 | RXRβ-PA-GRIP-1 | RXRγ-PA-GRIP-1 |
---|---|---|---|
PDB codes | 7A77 | 7A78 | 7A79 |
Beamline | DESY P13 | SLS X06SA | SLS X06SA |
Data Collection | |||
Resolution b (Å) | 43.10–1.50 (1.55–1.50) | 45.0–1.72 (1.78–1.72) | 46.01–2.05 (2.12–2.05) |
Space group | P 43212 | P 43212 | P 212121 |
Cell dimensions | a=b=66.2, c=110.6 Å | a=b=63.6, c=110.1 Å | a=63.3, b=67.1, c=110.7 Å |
α=β=γ=90.0 | α=β=γ=90.0 | α=β=γ=90.0° | |
Number of unique reflections | 40,177 (3855) | 24,821 (2373) | 30,235 (2910) |
Completeness (%) | 100.0 (100.0) | 100.0 (100.0) | 99.9 (100.0) |
I/σI | 20.0 (4.0) | 15.3 (2.3) | 8.2 (2.0) |
Rmerge (%) | 0.073 (0.703) | 0.059 (0.875) | 0.148 (0.782) |
CC (1/2) | 0.999 (0.876) | 0.999 (0.769) | 0.994 (0.754) |
Redundancy | 15.6 (14.2) | 8.0 (8.3) | 8.5 (7.9) |
Refinement | |||
Number atoms in refinement (P/G/L/O) c | 1841/ 99/ 18/ 237 | 1688/ 111/ 18/ 160 | 3416/ 210/ 36/ 182 |
B factor (P/G/L/O) c (Å2) | 22/ 29/ 29/ 35 | 37/ 40/ 39/ 44 | 38/ 48/ 47/ 43 |
Rfact (%) | 15.2 | 17.7 | 19.5 |
Rfree (%) | 18.0 | 20.4 | 24.8 |
rms deviation bond c (Å) | 0.015 | 0.013 | 0.014 |
rms deviation angle c (°) | 1.6 | 1.4 | 1.4 |
Molprobity Ramachandran | |||
Favor (%) | 97.4 | 99.0 | 98.0 |
Outlier (%) | 0 | 0 | 0 |
Crystallization condition d | 17% PEG 3350, 0.2 M ammonium acetate, 0.1 M tris, pH 8.0 | 16% low-molecular-weight PEG smears, 0.1 M HEPES, pH 7.5, 5% ethylene glycol, 0.1 M KCl | 17% PEG 3350, 0.1 M potassium citrate |
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Chaikuad, A.; Pollinger, J.; Rühl, M.; Ni, X.; Kilu, W.; Heering, J.; Merk, D. Comprehensive Set of Tertiary Complex Structures and Palmitic Acid Binding Provide Molecular Insights into Ligand Design for RXR Isoforms. Int. J. Mol. Sci. 2020, 21, 8457. https://doi.org/10.3390/ijms21228457
Chaikuad A, Pollinger J, Rühl M, Ni X, Kilu W, Heering J, Merk D. Comprehensive Set of Tertiary Complex Structures and Palmitic Acid Binding Provide Molecular Insights into Ligand Design for RXR Isoforms. International Journal of Molecular Sciences. 2020; 21(22):8457. https://doi.org/10.3390/ijms21228457
Chicago/Turabian StyleChaikuad, Apirat, Julius Pollinger, Michael Rühl, Xiaomin Ni, Whitney Kilu, Jan Heering, and Daniel Merk. 2020. "Comprehensive Set of Tertiary Complex Structures and Palmitic Acid Binding Provide Molecular Insights into Ligand Design for RXR Isoforms" International Journal of Molecular Sciences 21, no. 22: 8457. https://doi.org/10.3390/ijms21228457