ABSTRACT
The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.
