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结构域包含蛋白在细胞信号中的功能。BRCT基序最初在乳腺癌蛋白BRCA1中被鉴定为串联对，后来在参与DNA修复和细胞周期控制的其他蛋白质中被鉴定为串联对。我们证明了BRCT结构域是一个磷蛋白结合基序。BRCA1利用BRCT串联重复序列，而其他蛋白质，包括拓扑异构酶II结合蛋白TopBP1和DNA连接酶IIIpha，通过BRCT结构域的同源二聚体识别它们的磷酸化靶标。也有强有力的证据表明，NBS1(奈梅根断裂综合征)蛋白通过一系列BRCT和FHA结构域与磷酸化组蛋白H_2AX结合。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同源二聚体、BRCT-FHA串联结构域--与磷酸化靶标结合，这表明至少存在三种模式的磷蛋白识别。目前还没有BRCT/磷蛋白复合体的3D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光谱、微量热学和生物化学技术来确定3D结构，并探索说明所有三种BRCT结合模式的蛋白质-磷肽复合物相互作用的能量学。我们还将测试磷酸肽结合调节BRCT介导的其他相互作用的假设。我们的具体目标是： 目的1.探讨BRCA1与解旋酶BACH1和CtIP辅阻遏子磷酸化多肽的相互作用，阐明BRCT串联重复序列识别磷蛋白的分子机制。 目的2.探讨TopBP1与E2F1转录因子磷酸化多肽的相互作用，阐明BRCT同源二聚体识别磷蛋白的分子机制。 目的3.探讨NBS1与磷酸化组蛋白H_2AX多肽的相互作用，阐明FHA-BRCT串联结构域识别磷蛋白的分子机制。 目的4.研究磷酸蛋白与DNA连接酶IIIa结合对其BRCT与DNA修复蛋白XRCC1相互作用的可能调节作用。

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.