Mechanisms, Structure, and Regulation of CFTR's NBD's

描述（由申请人提供）：CFTR（囊性纤维化跨膜传导调节因子）由CF患者中突变的基因编码，是约50种人ATP结合盒（ABC）蛋白之一，属于ABC-C亚家族，该亚家族还包括SUR（磺酰脲受体）和MRP（多药耐药相关）蛋白。与其他ABC蛋白不同，CFTR是一种离子通道;它允许跨上皮液体运动所需的Cl-流动。CFTR通道孔的打开和关闭由ATP与CFTR的两个核苷酸结合结构域（NBD）结合和ATP水解控制。这些NBD事件到通道门的转导通过CFTR的调节（R）结构域中的多个丝氨酸的cAMP依赖性蛋白激酶的磷酸化来调节。该研究的目的是从分子水平上了解NBD功能和通道门控的调控机制。了解控制CFTR通道开放和关闭的精确机制可能有助于CF患者中由于突变CFTR通道表达而导致离子流不足的细胞的药理学拯救;这包括到达细胞表面的数量不足的突变体，孔电导降低的突变体，以及开放时间不足的突变体。具体目标基本不变。第一个解决了NBD的外观，它们如何工作，它们如何相互作用，以及它们如何控制通道的门。工作假设是CFTR的两个NBD在结构上不同（ABC-C家族成员的特征），在ATP结合时，它们形成头-尾二聚体，所述头-尾二聚体将两个ATP分子包封在二聚体界面内的复合催化位点中，二聚化驱动通道打开，并且ATP在NBD 2催化位点处的水解促使通道关闭; ATP在NBD 1催化位点保持结合数分钟而不被水解。第二个目的是解决磷酸化（以及在哪个或哪些位点）如何允许通道开放，以及额外的磷酸化如何促进通道开放状态的稳定。野生型和突变CFTR通道将在卵母细胞和哺乳动物细胞中表达，并使用生物物理学、电生理学和生物化学方法分析其结构和功能。单通道门控动力学的突变周期测量将探测CFTR的残基和结构域之间的能量相互作用。光标记将探测核苷酸与NBD的相互作用。原核NBD异二聚体的结构分析，与CFTR中的活性和死催化位点，将阐明CFTR的NBD的机制。

DESCRIPTION (provided by applicant): CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF patients, is one of approximately 50 human ATP-binding cassette (ABC) proteins, and belongs to subfamily ABC-C which also includes SUR (sulfonylurea receptor) and MRP (multidrug resistance related) proteins. Unlike other ABC proteins, CFTR is an ion channel; it allows the Cl- flow needed for transepithelial fluid movement. Opening and closing of the CFTR channel pore are controlled by ATP binding to CFTR's two nucleotide binding domains (NBDs) and by ATP hydrolysis. Transduction of these NBD events to the channel gates is regulated by phosphorylation, by cAMP-dependent protein kinase, of multiple serines in CFTR's regulatory (R) domain. The goal of the proposed research is to understand, in molecular detail, the mechanisms regulating NBD function and channel gating. Knowing the precise mechanisms that control CFTR channel opening and closing might help pharmacological rescue in CF patients of cells with inadequate ion flow due to expression of mutant CFTR channels; this includes mutants that reach the cell surface in inadequate numbers, those with diminished pore conductance, and those that spend an insufficient time open. The specific aims are essentially unchanged. The first addresses what the NBDs look like, how they function, how they interact, and how they control the channel's gates. The working hypothesis is that CFTR's two NBDs are structurally dissimilar (a characteristic of ABC-C family members), that upon ATP binding they form head-to-tail dimers that enclose two ATP molecules in composite catalytic sites within the dimer interface, that the dimerization drives channel opening, and that hydrolysis of the ATP at the NBD2 catalytic site prompts channel closing; ATP remains bound at the NBD1 catalytic site for several minutes without being hydrolyzed. The second aim addresses how phosphorylation (and at which site or sites) permits channel opening, and how additional phosphorylation promotes stabilization of the channel open state. Wild-type and mutant CFTR channels will be expressed in oocytes and mammalian cells, and their structure and function analyzed using biophysical, electrophysiological, and biochemical methods. Mutant cycle measurements of single-channel gating kinetics will probe energetic interactions between residues and domains of CFTR. Photolabeling will probe nucleotide interactions with the NBDs. Structural analysis of prokaryotic NBD heterodimers, with an active and a dead catalytic site as in CFTR, will elucidate mechanisms in CFTR's NBDs.