Mechanisms, Structure, and Regulation of CFTRs NBDs

描述（由申请人提供）：CFTR（囊性纤维化跨膜传导调节因子）由CF患者突变的基因编码，是48种人类atp结合盒（ABC）蛋白之一。CFTR与其他医学上重要的蛋白MRP（多药耐药相关蛋白）和SUR（磺酰脲受体）一样，属于ABC-C亚家族。但与其他ABC蛋白不同的是，CFTR是一个离子通道，可以进行高分辨率的功能测试。这些实验的证据表明，在两个细胞质核苷酸结合结构域（NBDs）之间的界面上，由ATP结合和水解驱动的构象变化循环，在大多数ABC蛋白中传递到跨膜结构域以驱动底物运输，在CFTR中打开和关闭通道门。这种门控调节了快速下坡的阴离子流动，这是上皮液体运动所必需的。CFTR可以被认为是一个从ABC祖先进化而来的破损转运蛋白，它的一个门失去了完整性。本研究的目标是从分子层面详细了解CFTR的NBD的结构和功能机制，它们之间的相互作用，跨膜结构域（TMDs）通道门的转导途径，以及NBD功能和通道门的调节机制。了解控制CFTR Cl-通道打开和关闭的精确机制可能有助于CF患者未来的药物救援，这些CF患者的患病细胞由于CFTR通道突变的表达而导致离子流动不足，包括那些不能达到足够数量的细胞表面的细胞，那些单通道电导减少的细胞，以及那些开放时间不足的细胞。具体目标是：(1)加强和完善我们现有的CFTR通道门控周期模型；(2)精确定位伴随通道门控的两个nbd之间以及tmd中nbd与通道门之间的动态重排位置；(3)确定CFTR在浇注周期内发生的结构运动程度。野生型和突变型CFTR通道将在卵母细胞中表达，并使用电生理、生物物理和生化方法分析其结构和功能。单通道门控动力学的测量将测试门控循环模型。引入的目标半胱氨酸对单功能和双功能硫醇特异性试剂的可及性的实时门态依赖将探测CFTR残基和结构域之间的相互作用。对具有一个活性和一个残缺复合催化位点的超嗜热细菌的不对称ABC蛋白进行结构分析，将阐明CFTR的分子转导机制。

DESCRIPTION (provided by applicant): CFTR (cystic fibrosis transmembrane conductance regulator), encoded by the gene mutated in CF patients, is one of 48 human ATP-binding cassette (ABC) proteins. CFTR belongs to subfamily ABC-C, like other medically important proteins MRP (multidrug resistance related protein) and SUR (sulfonylurea receptor). But unlike any other ABC protein, CFTR is an ion channel, allowing high-resolution tests of function. Evidence from such tests suggests that the same cycle of conformational changes, driven by ATP binding and hydrolysis in the interface between the two cytoplasmic nucleotide-binding domains (NBDs), that in most ABC proteins is transmitted to the transmembrane domains to power substrate transport, in CFTR opens and closes the channel gate. This gating regulates the rapid downhill anion flow needed for transepithelial fluid movement. CFTR can be considered to be a broken transporter that evolved from an ABC ancestor by loss of integrity of one of its gates. The goal of the proposed research remains to understand, in molecular detail, the structure and mechanisms of function of CFTR's NBDs, the interactions between them, the transduction pathway to the channel's gate in the transmembrane domains (TMDs), and the mechanisms by which NBD function and channel gating are regulated. Understanding the precise mechanisms that control opening and closing of CFTR Cl- channels might aid future pharmacological rescue in CF patients of diseased cells with inadequate ion flow due to expression of mutant CFTR channels, including those that fail to reach the cell surface in adequate numbers, those with diminished single-channel conductance, and those that spend an insufficient fraction of the time open. The Specific Aims are: (1) to strengthen and refine our present model of the CFTR channel gating cycle; (2) to pinpoint the locations of the dynamic rearrangements between the two NBDs, and between the NBDs and the channel gates in the TMDs, that accompany channel gating; and (3) to determine the extents of structural motions that occur within CFTR during its gating cycle. Wild-type and mutant CFTR channels will be expressed in oocytes and their structure and function will be analyzed using electrophysiological, biophysical, and biochemical methods. Measurements of single-channel gating kinetics will test gating cycle models. Real-time gating-state dependence of accessibility of introduced target cysteines to monofunctional and bifunctional thiol-specific reagents will probe interactions between residues and domains of CFTR. Structural analysis of asymmetric ABC proteins from hyperthermophiles, with one active and one crippled composite catalytic site, will elucidate molecular transduction mechanisms in CFTR. PUBLIC HEALTH RELEVANCE: Cystic fibrosis, the most common lethal genetic disease in the US, is caused by defects in a single protein (called CFTR) that normally helps move salt across the surfaces of cells in the lungs, intestines, pancreas, and sweat ducts. Our research aims to understand exactly how a CFTR protein usually works, to help doctors better choose ways to make up the deficit caused by poorly performing CFTR proteins in cystic fibrosis patients.