Structure Function of FHA Domain in Signaling and Cancer

描述（申请人摘要）：本提案的目的是 表征一类新的磷蛋白的结构和特异性 结合域，“叉头相关域”（FHA域），从四个 重要的蛋白质：人Chk 2、人Nitrifos、人Ki 67和酵母Rad 53。所有 这些蛋白质（Ki 67除外）参与DNA损伤的信号传导事件 这三种人类蛋白质的功能直接关系到 癌该项目包括五个具体目标，涉及两个 合作者（L-J. Byeon和D. Pei）。具体目标1是表达不同的 FHA结构域，并通过NMR确定其三级结构。具体目标2是 为了对FHA结构域的配体特异性进行严格分析， 含有pTyr、pSer或pThr的组合肽文库在 结合物理方法（质谱、表面等离子体 共振、荧光共振能量转移和NMR）。具体目标3是 为了确定FHA结构域与所述结构域的复合物的结构， 在先前的特定目的中通过NMR鉴定的紧密结合的磷酸肽。 具体目标4是对结构-功能进行定量分析 FHA域的关系。在综合体结构的基础上， 主要研究者将突变FHA结构域中的关键识别残基 并确定结合亲和力的变化。此外，他将努力 设计新的磷酸肽特异性。目标是充分了解 不同FHA结构域的定量结构-功能关系。 具体目标5是鉴定在细胞表面的Rad 9的天然FHA结合位点。 目标2中鉴定的最佳肽序列的基础。的组合 定点诱变、结合测定和肽图谱将被 就业。总的来说，所产生的信息将有助于了解 PHA结构域在与DNA相关的细胞调控中的化学机制 损伤和癌症。

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.