Characterization of two synthetic ligands of peroxisome proliferator-activated receptor γ (PPARγ) by cofactor recruitment, site-directed mutagenesis and structure analysis

In this study we report the behavior of two PPAR enantiomeric ligands (R-1 and S-1). Cell-based reporter assays indicate that both enantiomers are dual PPAR/ ligands, being R-1 a full agonist of PPAR and and S-1 a partial agonist of PPAR. 3D-structure analysis of the PPAR ligand binding domain in the complex with the two ligands shows that the suboptimal conformation of helix 12 in the PPAR/S-1 complex is the consequence of a steric hindrance between the ethyl group of the ligand and the Q286 in helix 3. Site-directed mutagenesis confirms that Q286 is a key residue in determining the activity of different ligands. By using FRET assays, we found that the coactivators SRC-1, PGC-1, RIP140, TIF-2 are recruited by R-1, S-1 and rosiglitazone with similar EC50, whereas CBP affinity is higher in the presence of rosiglitazone. Conversely, only S-1 allows the association of the corepressor N-CoR to PPAR as opposed to rosiglitazone and R-1, providing a functional explanation to the partial agonist behavior of S-1. ChIP assays confirmed that S-1 enhances the association of N-CoR to PPAR target promoters in the native chromatin context, whereas rosiglitazone and R-1 recruit less N-CoR. Altogether, our results provide a rationale for the distinct behavior of different PPAR ligands. Supported by CARIPLO Foundation, Harvard-Armenise Foundation