Molecular Mechanisms of CFTR Regulation

描述（由申请人提供）：囊性纤维化跨膜传导调节因子（CFTR）是一种ATP调节的氯离子通道，决定上皮细胞顶膜中电解质和液体转运的速率。CFTR功能异常与囊性纤维化和分泌性腹泻的发病机制有关。我们的长期目标是在原子水平上了解CFTR调节的分子机制，并开发新的策略来调节该通道的活性和治疗CFTR相关疾病。CFTR拓扑结构由两个跨膜结构域和五个胞质结构域组成：N-末端结构域（NTD），两个核苷酸结合结构域，调节结构域（R）和C-末端结构域（CTD）。CFTR活性通过R结构域的磷酸化、NBD的ATP水解以及其NTD和CTD结构域分别与突触融合蛋白1A和NHERF蛋白的相互作用来调节。然而，调控机制仍然未知，主要是因为CFTR结构域的三维结构及其与细胞内调控蛋白相互作用的结构基础仍然难以捉摸。该提案解决了这些问题，并侧重于细胞质CFTR结构域及其与调控蛋白的复合物的结构分析，使用分子生物学技术和X射线晶体学。具体目标是： 1.剖析通过CFTR CTD与NHERF PDZ 1和PDZ 2结构域的相互作用介导的CFTR通道门控的结构基础。 2.阐明CFTR NTD与syntaxin 1A相互作用调节CFTR通道活性的分子机制。 3.为了确定CFTR NBD 1和NBD 2结构域的三维原子结构。 这些研究将首次提供四个胞质CFTR结构域的高分辨率三维结构，以及蛋白质syntaxin 1A和NHERF调控CFTR的结构基础。这些信息是阐明控制CFTR通道门控的基本分子机制的重要一步。 重要的是，这些复合物的原子坐标可用于基于结构的合理设计药物，这些药物将选择性地改变CFTR活性，临床应用于治疗囊性纤维化和分泌性腹泻。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated chloride channel that determines the rate of electrolyte and fluid transport in the apical membrane of epithelial cells. Abnormal CFTR function is associated with the pathogenesis of cystic fibrosis and secretory diarrhea. Our long-term objective is to understand the molecular mechanisms underlying the regulation of CFTR at the atomic level and develop novel strategies for modulating the activity of this channel and treating the CFTR-associated diseases. The CFTR topology consists of two membrane-spanning domains and five cytoplasmic domains: an N-terminal domain (NTD), two nucleotide-binding domains, a regulatory domain (R) and a C-terminal domain (CTD). The CFTR activity is modulated through phosphorylation of the R domain, ATP hydrolysis by the NBDs, and interactions of its NTD and CTD domains with syntaxin 1A and NHERF proteins, respectively. However the regulatory mechanisms remain unknown primarily because the three-dimensional structure of the CFTR domains and the structural basis of their interaction with intracellular regulatory proteins remain elusive. This proposal addresses these questions and focuses on the structural analysis of cytoplasmic CFTR domains and their complexes with regulatory proteins, using molecular biology techniques and X-ray crystallography. The specific aims are: 1. To dissect the structural basis of CFTR channel gating mediated through the interaction of the CFTR CTD with the NHERF PDZ1 and PDZ2 domains. 2. To elucidate the molecular mechanisms underlying the regulation of CFTR channel activity through the interaction of the CFTR NTD with syntaxin 1A. 3. To determine the three-dimensional atomic structures of the CFTR NBD1 and NBD2 domains. These studies will provide the first high-resolution three-dimensional structures of four cytoplasmic CFTR domains and the structural basis of CFTR regulation by proteins syntaxin 1A and NHERF. This information is an essential step towards elucidating the basic molecular mechanisms that control the CFTR channel gating. Importantly, the atomic coordinates of these complexes could be used for structure-based rational design of drugs that would modify selectively the CFTR activity with clinical applications in the treatment of cystic fibrosis and secretory diarrhea.