Structure Function of FHA Domain in Signaling and Cancer

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DESCRIPTION (Applicant's abstract): The objective of this proposal is to characterize the structure and specificity of a new class of phosphoprotein binding domain, the "forkhead-associated domain" (FHA domain), from four important proteins: human Chk2, human Nibrin, human Ki67, and yeast Rad53. All these proteins (except Ki67) are involved in signaling events in the DNA damage response, and the functions of the three human proteins are directly related to cancer. The project consists of five specific aims and involves two collaborators (L-J. Byeon and D. Pei). Specific Aim 1 is to express different FHA domains and determine their tertiary structures by NMR. Specific Aim 2 is to perform rigorous analyses of the ligand specificity of the FHA domains by use of combinatorial peptide libraries containing pTyr, pSer, or pThr, in combination with physical methods (mass spectrometry, surface plasmon resonance, fluorescence resonance energy transfer, and NMR). Specific Aim 3 is to determine the structures of the complexes of the FHA domain with the tight-binding phosphopeptides identified in the previous specific aim by NMR. Specific Aim 4 is to perform quantitative analyses of the structure-function relationship of FHA domains. On the basis of the structures of the complex, the principal investigator will mutate key recognition residues in the FHA domain and determine the changes in the binding affinity. Furthermore, he will try to engineer new phosphopeptide specificity. The goal is to fully understand the quantitative structure-function relationship for different FHA domains. Specific Aim 5 is to identify the natural FHA-binding site of Rad9 on the basis of the optimal peptide sequences identified in Aim 2. A combination of site-directed mutagenesis, binding assays, and peptide mapping will be employed. Overall, the information generated will lead to an understanding of the chemical mechanism of PHA domains in cellular controls related to DNA damage and cancer.