Peroxisome proliferator-activated receptor α interacts with high affinity and is conformationally responsive to endogenous ligands

Abstract

Although the peroxisome proliferator-activated receptor (PPARα) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Like-wise, it is not known how putative endogenous ligands alter PPARα conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPARα exhibits high affinity (1-14 nM Kd values) for unsaturated long chain fatty acyl-CoAs as well as un saturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPARα aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25-29 Å, confirming close molecular interaction. Interestingly, although PPARα also exhibited high affinity for saturated long chain fatty acyl-CoAs, regardless of chain length (1-13 nM Kd values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPARα for fatty acyl-CoAs and unsaturated fatty acids, CoA thioesters of peroxisome proliferator drugs were bound with 5-6-fold higher affinities than their free acid forms. Circular clichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weals affinity ligands (saturated fatty acids), elicited conformational changes in PPARα structure, a hallmark of ligand-activated nuclear receptors. Finally, these ligand specificities and induced conformational changes correlated functionally with co-activator binding. In summary, since nuclear concentrations of these ligands are in the nanomolar range, long chain fatty acyl-CoAs and unsaturated fatty acids may both represent endogenous PPAKα ligands. Furthermore, the finding that saturated fatty acyl-CoAs, rather than saturated fatty acids, are high affinity PPARα ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.