Effect of heterodimer partner RXRα on PPARγ activation function-2 helix in solution

Abstract

The structural mechanism of allosteric communication between retinoid X receptor (RXR) and its heterodimer partners remains controversial. As a first step towards addressing this question, we report a nuclear magnetic resonance (NMR) study on the GW1929-bound peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding domain (LBD) with and without the 9-cis-retinoic acid (9cRA)-bound RXRα LBD. Sequence-specific 13 C α , 13 C β and 13 CO resonance assignments have been established for over 95% of the 275 residues in the PPARγ LBD monomer. The 1 HN, 15 N and 13 CO chemical shift perturbations induced by the RXR LBD binding are located at not only the heterodimer interface that includes the C-terminal residue Y477, but also residues Y473 and K474 in the activation function-2 (AF-2) helix. This result suggests that 9cRA-bound RXRα can affect the PPARγ AF-2 helix in solution, and demonstrates that NMR is a powerful new tool for studying the mechanism of allosteric ligand activation in RXR heterodimers.

The RXRα C-terminus T462 is a NMR sensor for coactivator peptide binding

“…It is important to determine whether this phenomenon can be extended to a RXR heterodimer. We recently demonstrated that it is feasible to monitor residue-specific structural and dynamic changes of a RXR LBD heterodimer in response to ligand binding in solution [15].…”

“…Our previous high-resolution nuclear magnetic resonance (NMR) study [5] suggested that 9cRA binding stabilized the ligand-binding pocket and had an effect on the dimer interface. Surprisingly, the AF-2 helix only exhibited a moderate chemical shift perturbation and little changes in 15 N relaxation parameters induced by 9cRA binding.…”

“…A two-fold molar excess of the peptide over the LBD was added to the sample following 9cRA addition, and another one molar equivalent amount of the peptide was added to the sample prior to the final exchange. For NMR titration, 10, 25 or 100 mM peptide-DMSO-d 6 stock solution was added to 0.3-0.4 mM U-[ 2 H, 15 N]-enriched RXRα LBD-9cRA binary complex (the holo-LBD) stock solution. The final 500-μl-NMR samples contained 0.27 mM binary complex and 0.0, 0.14, 0.27, 0.40, 0.54, 1.5, 3.0, or 5.0 mM GRIP1 peptide, respectively.…”

“…All experiments employed the transverse relaxation-optimized spectroscopy (TROSY) scheme [8] unless specified otherwise. For sequence-specific resonance assignments and backbone 15 N relaxation measurements, data collection, processing and analysis were as described [5]. For NMR titration, the 2-dimensional (2D) 1 H-15 N heteronuclear single quantum correlation (HSQC) spectra were recorded on the 700-MHz spectrometer, with 10500 and 1983 Hz spectral width and 672 and 116 complex data points in the 1 H and 15 N dimension, respectively, 64 scans per free induction decay, 1.8 s relaxation delay, and a total of 8 h for each spectrum.…”

“…Our results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility. 15 N, 13 C]-enriched RXRα LBDs were expressed in Escherichia coli BL21-DE3 bacteria, isolated by Talon affinity chromatography, digested by thrombin to remove the N-terminal His 6 tag, and finally purified by gel filtration chromatography as described [5].…”