Structural Basis of Cell Signaling by BRCT Domains

描述（由申请人提供）：我们的长期目标是了解BRCT结构域蛋白在细胞信号传导中的功能。BRCT基序首先在乳腺癌蛋白BRCA1中被鉴定为串联对，后来在其他参与DNA修复和细胞周期控制的蛋白中也被鉴定为串联对。我们发现BRCT结构域是一个磷酸化蛋白结合基序。虽然BRCA1利用BRCT串联重复，但其他蛋白质，包括拓扑异构酶II结合蛋白，TopBP1和DNA连接酶iii α，通过BRCT结构域的同型二聚体识别其磷酸化靶标。也有强有力的证据表明NBS1（奈梅根断裂综合征）蛋白通过BRCT和FHA结构域串联结合磷酸化组蛋白H2AX。这些观察结果表明，BRCT蛋白通过不同耦合的BRCT结构域（即BRCT串联重复序列、BRCT同型二聚体、BRCT- fha串联结构域）结合磷酸化靶点，表明存在至少3种磷酸化蛋白识别模式。没有BRCT/磷酸蛋白复合物的三维结构。为了了解磷蛋白识别是如何实现的，我们将使用核磁共振波谱，微量热法和生物化学技术来确定三维结构，并探测蛋白质-磷酸肽复合物相互作用的能量学，说明所有3种BRCT结合模式。我们还将验证磷酸肽结合调节其他brct介导的相互作用的假设。我们的具体目标是：

DESCRIPTION (provided by applicant): Our long-term goal is to understand the function of BRCT domain-containing proteins in cellular signaling. The BRCT motif was first identified as a tandem pair in the breast cancer protein BRCA1, and later in other proteins involved in DNA repair and cell cycle control. We showed that the BRCT domain is a phosphoprotein binding motif. While BRCA1 utilizes a BRCT tandem repeat, other proteins, including topoisomerase II binding protein, TopBP1, and DNA ligase IIIalpha, recognize their phosphorylated targets by means of a homodimer of BRCT domains. There is also strong evidence that NBS1 (Nijmegen Breakage Syndrome) protein binds phosphorylated histone H2AX through a tandem of BRCT and FHA domains. These observations that BRCT proteins bind phosphorylated targets through differently coupled BRCT domains - i.e., BRCT tandem repeats, BRCT homodimer, BRCT-FHA tandem domains - indicate that at least 3 modes of phosphoprotein recognition exist. There is no 3D structure of a BRCT/phosphoprotein complex. To understand how phosphoprotein recognition is achieved, we will use NMR spectroscopy, microcalorimetry and biochemistry techniques to determine the 3D structures, and probe the energetics of interaction of protein-phosphopeptide complexes illustrating all 3 BRCT binding modes. We will also test the hypothesis that phosphopeptide binding modulates other BRCT-mediated interactions. Our specific aims are: Aim 1. Probe the interaction of BRCA1 with phosphorylated peptides from the helicase BACH1 and CtIP corepressor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT tandem repeat. Aim 2. Probe the interaction between TopBP1 and a phosphorylated peptide from E2F1 transcription factor to elucidate the molecular mechanism of phosphoprotein recognition by a BRCT homodimer. Aim 3. Probe the interaction of NBS1 with a phosphorylated histone H2AX peptide to elucidate the molecular mechanism of phosphoprotein recognition by FHA-BRCT tandem domains. Aim 4. Investigate a possible regulatory effect of phosphoprotein binding to DNA ligase IIIa on its BRCT-mediated interaction with DNA repair protein XRCC1.