ADIPOCYTE LIPID-BINDING PROTEIN COMPLEXED WITH ARACHIDONIC-ACID - TITRATION CALORIMETRY AND X-RAY CRYSTALLOGRAPHIC STUDIES

The association of the adipocyte lipid-binding protein (ALBP) with ara chidonic acid (all cis, 20:4 Delta(5,8,11,14)) and oleic acid (cis, 18 :1 Delta(9)) has been examined by titration calorimentry. In addition, the crystal structure of ALBP with bound arachidonic acid has also be en obtained. Crystallographic analysis of the arachidonic acid.ALBP co mplex along with the previously reported oleic acid-ALBP structure (Xu , Z., Bernlohr, D. A., and Banaszak, L. J. (1993) J. Biol. Chem. 268, 7874-7884) provides a framework for the molecular examination of prote in-lipid association. Isothermal titration calorimetry revealed high a ffinity association of both unsaturated fatty acids with the protein. The calorimetric data yielded the following thermodynamic parameters f or arachidonic acid: K-d= 4.4 mu M, n = 0.8, Delta G = -7370 cal/mol, Delta H = -6770 cal/mol, and T Delta S = +600 cal/mol. For oleic acid, the thermodynamic parameters were K-d = 2.4 mu M, n = 0.9, Delta G = -7770 cal/mol, Delta H = -6050 cal/mol, and T Delta S = +1720 cal/mol. The identification of thermodynamically dominating enthalpic factors for both fatty acids are consistent with the crystallographic studies demonstrating the interaction of the fatty acid carboxylate with a com bination of Arg(106), Arg(126), and Tyr(128). The crystallographic ref inement of the protein-arachidonate complex was carried out to 1.6 Ang strom with the resultant R factor of 0.19. Within the cavity of the cr ystalline binding protein, the arachidonate was found in a hairpin con formation. The conformation of the bound ligand is consistent with acc eptable torsional angles and the four cis double bonds in arachidonate . These results demonstrate that arachidonate is a ligand for ALBP. Th ey provide thermodynamic and structural data concerning the physical b asis for protein-lipid interaction and suggest that intracellular lipi d-binding proteins may mediate the biological effects of polyunsaturat ed fatty acids in vivo.