Structure Function of FHA Domain in Signaling and Cancer

说明(申请人摘要)：本提案的目的是 一类新的磷蛋白的结构和特异性的表征 结合结构域，“叉头相关结构域”(FHA结构域)，来自四个 重要蛋白质：人Chk2、人Nibrin、人Ki67和酵母Rad53。全 这些蛋白质(Ki67除外)参与了dna损伤的信号传递。 而这三种人类蛋白质的功能直接关系到 癌症。该项目由五个具体目标组成，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，并通过核磁共振确定其三级结构。具体目标2是 通过对FHA结构域的配基特异性进行严格的分析 使用包含pTyr、pSer或pThr的组合肽库 与物理方法相结合(质谱仪、表面等离子激元 共振、荧光共振能量转移和核磁共振)。具体目标3是 确定FHA结构域与 用核磁共振方法鉴定了前一特定目的中的紧密结合的磷酸肽。 具体目标4是执行结构-功能的定量分析 FHA结构域之间的关系。根据复合体的结构， 首席研究员将突变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 设计新的磷酸多肽专一性。我们的目标是充分了解 不同FHA结构域的定量结构-功能关系。 具体目的5是确定RAD9的天然FHA结合部位 目标2中确定的最佳多肽序列的基础。 定点突变、结合分析和多肽图谱将是 受雇的。总体而言，生成的信息将导致对 与DNA相关的细胞调控中PHA结构域的化学机制 损害和癌症。

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.