Adipocyte lipid binding protein mutations: In search of binding specificity determinants

The beta-barrel intracellular lipid binding proteins (iLBPs) have been the focus of much research to understand the roles of ligand acquisition, affinity, specificity, and cavity location in ligand transport. The adipocyte lipid binding protein (ALBP) is a broadly studied member of this family. It is likely that a set of specific amino acids from the mouth and pocket of the binding cavity play a role in directing ligand binding. To test this hypothesis, a series of ALBP mutations were designed to assess the importance of specific residues in ligand binding. A mutation in the binding cavity (E72K) was made to disrupt the internal ordered water molecule structure. Crystal structures of this mutation were solved with and without bound oleic acid at 2.2A and 2.3A respectively. Current refinement of this mutation is inconclusive as to the affect on the water network. A mutation (I73E/A77V/D77G) was made to test the betaE-betaD loop region near the mouth of the binding pocket. Crystal structures of this mutation were solved with and without bound oleic acid bound at 1.5A and 1.7A respectively. Stability, isothermal titration calorimetry, and fluorescent probe binding and displacement assays show that this mutant protein has a different thermodynamic profile and bound cavity position than the native form. A mutation (insertion of A and S after A36) was made to test the region where a helix-turn-helix motif hinges to the beta-barrel at the mouth of the pocket. Binding characterization of this mutation supports weaker ligand binding than the native form. Crystals of this mutation are being pursued. Four additional mutations (M20L, C117F, 1104W, and F27A/A28M/T29L) have been made to test their roles in ALBP binding and are ready for further study.