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

描述(申请人提供)：CFTR(囊性纤维化跨膜传导调节因子)，由CF患者突变的基因编码，是大约50个人类三磷酸腺苷结合盒(ABC)蛋白之一，属于ABC-C亚家族，还包括SUR(磺脲受体)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；它允许跨上皮液运动所需的氯离子流动。Cftr通道孔的打开和关闭由与cftr的两个核苷酸结合域(Nbd)的ATP结合和ATP的水解控制。这些NBD事件到通道门的转导是由cAMP依赖的蛋白激酶调节的，在CFTR的调节(R)结构域中的多个丝氨酸的磷酸化。这项拟议研究的目的是从分子上详细了解NBD功能和通道门控的调节机制。了解控制CFTR通道打开和关闭的精确机制可能有助于在药物上挽救由于突变的CFTR通道表达而导致离子流动不足的细胞；这包括数量不足到达细胞表面的突变体，孔电导降低的突变体，以及打开时间不足的突变体。具体目标基本上没有变化。第一个问题是NBD是什么样子的，它们是如何运作的，它们是如何相互作用的，以及它们是如何控制频道的大门的。工作假说是，CFTR的两个NBD在结构上是不同的(ABC-C家族成员的特征)，当与ATP结合时，它们形成头尾相连的二聚体，将两个ATP分子包裹在二聚体界面内的复合催化部位，二聚作用驱动通道开放，而Nbd2催化部位的ATP水解促使通道关闭；ATP在NBD1催化部位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。野生型和突变型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、电生理和生化方法分析其结构和功能。单通道门控动力学的突变循环测量将探索CFTR残基和结构域之间的能量相互作用。光标记将探测与NBD的核苷酸相互作用。对原核生物NBD异源二聚体的结构分析，与CFTR中一样，具有活性和死催化位，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.