Molecular Mechanisms of CFTR Function

描述(申请人提供)：囊性纤维化跨膜传导调节因子(CFTR)在脊椎动物上皮细胞盐分和液体动态平衡中起关键作用，它的缺失或功能障碍会导致人类囊性纤维化。在这个项目中，我们研究了CFTR单通道门控动力学，其结合和水解三磷酸腺苷的能力，以及其独特的R结构域的磷酸化状态控制。到目前为止的发现与一个模型是一致的，在该模型中，单体CFTR作为一个可水解配体门控通道，其中在ATP结合/水解和通道门控之间存在磷酸化调节的变构偶联。我们最近发现，磷酸化而不是影响ATP的水解，促进了未水解的ATP从NBD1中释放出来，并增加了大部分非结构的R结构域的旋转半径，这反过来又改变了膜跨结构域的构象。我们是唯一一个通过电子结晶学纯化、结晶并确定了完整蛋白质的低分辨率结构的小组。我们还通过计算生成了一个高分辨率模型，该模型满足大量已发表的实验数据，并揭示了我们通过半胱氨酸交联和结合实验证实的结构域相互作用。结构域交换相互作用已经被定义在分子相反半部分的细胞质和膜结构域之间，这对其组装和功能都是至关重要的。其中一个结构域交换作用是由苯丙氨酸残基508的芳香族侧链介导的，该侧链在大多数CF患者中被删除，我们独立地证明它直接参与通道门控。我们现在的主要目标是进一步阐明野生型CFTR的多个结构域在其正常功能中的作用以及它们之间的相互作用，然后确定这些结构域是如何被导致突变的主要囊性纤维化？F508改变的。第一个广泛目标将解决四个重大悬而未决的问题。第一个问题是，未水解的ATP从简并的签名基序Nbd2上解离，并被磷酸化促进从NBD1解离，是否有助于NBD之间界面的打开和通道的关闭。其次，我们将确定NBD和MSD之间的六个“传输接口”中的每一个的作用，包括那些调节分子相反两端之间的结构域交换或缠绕的接口。第三，将绘制跨膜结构域中螺旋间关系对通道激活刺激的响应变化，并确定它们对离子孔的贡献。第四，将确定NBDS和磷酸化控制的R结构域在CFTR功能过程中相互影响的情况。结合这些生化研究，电子结晶学将确定不同功能状态的更高分辨率的3D结构。在第二个主要目标中，由于我们将Phe508定位在3D结构中，我们将确定它的缺失对蛋白质其余部分的结构和功能的影响，以促进新治疗策略的开发。 与公共卫生相关：CFTR是一种独特的离子通道，它采用了一种经过修改的活性转运蛋白结构体系，当突变时会导致人类囊性纤维化。在几乎所有陆生和海洋脊椎动物中，磷酸化调节的通道为离子和液体在上皮表面的移动提供了一个限速步骤。因此，在这一功能的执行过程中了解其三维结构和动力学对于了解正常的人类健康和疾病具有重要的基础和实际意义。

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in vertebrate epithelial salt and fluid homeostasis and its absence or dysfunction results in cystic fibrosis in humans. In this project we have characterized CFTR single channel gating kinetics, its ability to bind and hydrolyze ATP, and its control by the phosphorylation state of its unique R domain. Findings thus far are consistent with a model in which monomeric CFTR acts as a hydrolysable-ligand gated channel in which there is phosphorylation regulated allosteric coupling between ATP binding/hydrolysis and channel gating. We have found recently that phosphorylation rather than influencing ATP hydrolysis, promotes release of unhydrolysed ATP from NBD1 and also increases the radius of gyration of the largely unstructured R domain which in turn alters the conformation of membrane spanning domains. We are the only group to have purified, crystallized, and determined a low resolution structure of the complete protein by electron crystallography. We have also generated a high resolution model computationally which satisfies a body of published experimental data and reveals domain interactions that we have confirmed by cysteine cross-linking and binding experiments. Domain-swapping interactions have been defined between cytoplasmic and membrane domains in opposite halves of the molecule which are crucial to both its assembly and function. One of these domain-swapping interactions is mediated by the aromatic side chain of phenylalanine residue 508, deleted in most CF patients, which we showed independently is directly involved in channel gating. Our major objectives now are to further elucidate the roles of wild-type CFTR's multiple domains and the interactions between them in its normal function and then to determine how these are altered by the major cystic fibrosis causing mutation, ?F508. The first broad aim will address four significant unresolved issues. The first asks whether unhydrolysed ATP disengagement from the degenerate signature motif of NBD2 and its phosphorylation stimulated dissociation from NBD1 contribute to the opening of the interface between the NBDs and the closing of the channel. Second, we will determine the role of each of the six "transmission interfaces" between the NBDs and MSDs including those that mediate the domain-swapping or intertwining between opposite sides of the molecule. Third, changes in inter-helical relationships in the membrane spanning domains in response to channel activating stimuli will be mapped and their contribution to the ion pore identified. Fourth, the influences of the NBDs and phosphorylation controlled R domain on each other during CFTR function will be determined. Higher resolution 3D structures of different functional states will be determined by electron crystallography in conjunction with these biochemical studies. In the second principal objective motivated by our localization of Phe508 in the 3D structure we will determine the impact of its absence on the structure and function of the rest of the protein in order to facilitate the development of new therapeutic strategies. PUBLIC HEALTH RELEVANCE: CFTR is a unique ion channel employing a modified active transporter structural architecture which when mutated results in cystic fibrosis in humans. The phosphorylation regulated channel provides a rate limiting step in ion and fluid movement across epithelial surfaces in virtually all terrestrial and marine vertebrates. Thus, knowledge of its 3D structure and dynamics during the performance of this function is of both fundamental and practical importance to understanding normal human health and disease.