Structural and functional characterization of the phosphorylated adipocyte lipid-binding protein (pp15).

A substrate for the insulin receptor kinase in 3T3-L1 adipocytes has previously been identified as the adipocyte lipid-binding protein (ALBP, also known as aP2 or p15). We have characterized the effect of tyrosyl phosphorylation on ALBP structure and ligand-binding properties. Phosphorylated ALBP (phospho-ALBP) was isolated by a combination of gel filtration, anion exchange chromatography, and immunoaffinity chromatography on anti-phosphotyrosine agarose. Circular dichroic spectroscopy indicated that the phosphoprotein was similar in structure to native ALBP. Phospho-ALBP exhibited a slight decrease in calculated alpha-helical content which was compensated for by an increase in beta-sheet structure. The wavelength yielding maximum tryptophan fluorescence was unaltered by phosphorylation (334 +/- 1 nm). However, the concentration of guanidine HCl yielding 50% denaturation was 1.43 M for ALBP and 0.92 M for phospho-ALBP. The delta Goapp was 3.87 and 3.25 kcal mol-1 for ALBP and phospho-ALBP, respectively, suggesting that phosphorylation destabilized the protein. To assess the binding characteristics of the phosphoprotein, a long-chain fatty acid affinity column was synthesized to which native ALBP specifically bound. In contrast, phospho-ALBP showed little or no affinity for the column. Furthermore, phosphorylation virtually abolished binding of the fluorescent fatty acid analogue 12-(9-anthroyloxy)oleic acid. Fatty acid binding activity was recovered (approximately 60%) upon dephosphorylation with protein tyrosine phosphatase. The structural studies, coupled with the crystal structure of the apoprotein, indicate that the dramatic reduction in binding affinity is likely a result of steric hindrance in the binding cavity or of electrostatic interactions of the phosphoryl group with the fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)