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

Although the peroxisome proliferator-activated receptor (PPAR alpha) binds and is activated by a variety of synthetic xenobiotics, the identity of the high affinity endogenous ligand(s) is incompletely resolved. Likewise, it is not known how putative endogenous ligands alter PPAR alpha conformation in order to affect transcriptional regulation. Direct fluorescence binding and fluorescence displacement assays showed for the first time that PPAR alpha exhibits high affinity (1-14 nM K(d) values) for unsaturated long chain fatty acyl-CoAs as well as unsaturated long chain fatty acids commonly found in mammalian cells. Fluorescence resonance energy transfer between PPAR alpha aromatic amino acids and bound corresponding naturally occurring fluorescent ligands (i.e. cis-parinaroyl-CoA, trans-parinaric acid) yielded intermolecular distances of 25-29 angstroms, confirming close molecular interaction. Interestingly, although PPAR alpha also exhibited high affinity for saturated long chain fatty acyl-CoAs, regardless of chain length (1-13 nM K(d) values), saturated long chain fatty acids were not significantly bound. In contrast to the similar affinities of PPAR alpha 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 dichroism demonstrated that high affinity ligands (long chain fatty acyl-CoAs, unsaturated fatty acids), but not weak affinity ligands (saturated fatty acids), elicited conformational changes in PPAR alpha 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 PPAR alpha ligands. Furthermore, the finding that saturated fatty acyl-CoAs, rather than saturated fatty acids, are high affinity PPAR alpha ligands provides a mechanism accounting for saturated fatty acid transactivation in cell-based assays.