Structure/Function Studies of Lipid Binding Proteins

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The central focus of this proposal is the establishment of a structural basis for biological recognition. Macromolecular recognition between two proteins is a combinatorial process bought about by the collective interactions of a variety of amino acids on a protein surface. Our studies are designed to address the issues of protein structure and the basis for protein-protein interaction. Two central issues will be addressed within this study: 1) What effects do small molecule ligands have upon protein structure and, 2) What domains on a protein surface are most critical for protein-protein interaction. Our experimental system revolves around the fatty acid binding protein of adipose cells, its interaction with fatty acid, and its phosphorylation by the insulin receptor. Fatty acid binding to the carrier protein allows for its tyrosyl phosphorylation by the insulin receptor. Phosphorylation of the holo fatty acid binding protein blocks fatty acid exchange and the release of the lipid from the binding domain. Dephosphorylation of the protein by a specific tyrosine phosphatase allows for ligand release and returns the fatty acid binding protein to the basal state. The association of the lipid binding protein with the insulin receptor kinase is thought to occur via interactions with both and acidic domain adjacent to the site of tyrosyl phosphorylation and a second domain, termed the SH2 domain, postulated to play a primary role in kinase recognition. We hypothesize that by a combinant of molecular and structural biology we will define these domains in protein-protein recognition. Specifically during the granting period we propose to examine molecular recognition by these two proteins by: 1. Determine the structure of the holo adipocyte fatty acid binding protein by X-ray crystallography. Examine the effect of the fatty acid on tyrosine-19 and on the SH2 domain. 2. Mutagenize the SH2 domain of ALBP. Create a dominant by generation of S101E, T103L and V118E. mutations. Examine the role of the SH2 domain in kinase recognition. Create a Y19F, dominant SH2 mutation, test as and inhibitor of the kinase. 3. Co-crystalize the Y19F, dominant SH2 ALBP with the soluble kinase domain of the receptor and ATP to form a ternary complex. Map the molecular interaction sites between the two proteins. The most significant aspects of this study will be the description at the molecular level how small molecules will affect protein structure and how that can be translated into differences in protein-protein recognition. For our particular system we will determine how the bound fatty acid influences the binding protein V and how that translates into a more favorable interaction with the insulin receptor kinase. Also we will be able, at the molecular level, to assess the relative contributions of acidic and SH2 domains on substrate proteins in tyrosine kinase interactiolns. In the long-term, the information gained here wil allow us to design specific in-situ tests of the role of the ALBP in lipid metabolism and its regulation be phosphorylation.