Ligand-induced stabilization of PPARgamma monitored by NMR spectroscopy: implications for nuclear receptor activation.

Nuclear receptors are ligand-dependent transcription factors that are mediators of the action of lipophilic hormones and other endogenous ligands and are the targets of drugs useful in a variety of therapeutic areas. Peroxisome proliferator-activated receptor (PPAR)gamma is a nuclear receptor that, acting as a heterodimer with RXR, mediates a variety of cellular effects including adipocyte-differentiation. Due to its role in modulating insulin sensitivity, it is the target of therapeutically active anti-diabetic agents such as rosiglitazone. We have assigned the chemical shifts of the backbone atoms of the 32 kDa ligand-binding domain of PPARgamma in the presence of bound rosiglitazone. Three-dimensional HNCO spectra of the apo ligand-binding domain (LBD) have less than half the expected number of cross-peaks. The missing cross-peaks are restored upon binding strong agonists such as rosiglitazone. The NMR results indicate that the apo-LBD of PPARgamma is in a conformationally mobile state, and that agonist binding is associated with a marked stabilization of the conformation. Mapping the missing peaks to the 3D X-ray crystallographic structure indicates the region of mobility is extensive and includes the ligand-binding region and the cofactor-binding site. This leads to the conclusion that activation of this nuclear receptor is a result of a population shift of a dynamic ensemble of conformations, rather than a two-state switch from an inactive to an active conformation. Our results have important implications for the mechanisms by which antagonists, partial agonists, and agonists of nuclear receptor function operate. Copyright 2000 Academic Press.