Characterization of the ADRP Fatty Acid Binding Site: A Fluorescence Binding and Circular Dichroic Study

Abstract

Although ADRP (adipose differentiation‐related protein, also known as perilipin 2) has been shown to bind fatty acids (FA) and other lipids with high affinity, the structure and location of the FA binding site has not been examined in detail. This is of some importance since ADRP's affinity for lipids and especially FA may be related to lipid droplet formation, triacylglycerol accumulation, and FA uptake and metabolism in the cell. The purpose of the present investigation was to characterize ADRP and several truncated mutants using fluorescence binding assays and circular dichroism (CD) to assess the structural requirements for binding. Results presented here show that removal of 120 amino acids from the N‐terminus, a high homology region common to other lipid droplet proteins increased the FA binding affinity (Kd) 43%. In contrast, deleting 254 residues decreased the Kd 24%. Removal of 173 amino acids from the C‐terminus, however did not significantly change the protein's ability to bind FA. Conformational changes upon binding were confirmed by CD. Taken together, these results indicate that residues 121–254 appear essential for highest affinity binding and show that the ability of ADRP to bind FA may depend on the proper folding of several domains, the partial deletion of which leads to altered, but not ablated ligand binding. This work was supported by NIH Grant DK70965 (BPA).