Molecular Mechanisms of CFTR Function

描述（由申请人提供）：囊性纤维化跨膜传导调节剂（CFTR）在脊椎动物上皮盐和液体稳态中起关键作用，其缺失或功能障碍导致人类囊性纤维化。在这个项目中，我们表征了CFTR单通道门控动力学，其结合和水解ATP的能力，以及其独特的R结构域磷酸化状态的控制。到目前为止，研究结果与单体CFTR作为水解配体门控通道的模型一致，其中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，磷酸化并没有影响ATP水解，而是促进了NBD1中未水解ATP的释放，并增加了大部分非结构化R结构域的旋转半径，从而改变了膜跨越结构域的构象。我们是唯一一个通过电子晶体学纯化、结晶和确定完整蛋白质低分辨率结构的团队。我们还通过计算生成了一个高分辨率模型，该模型满足了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在分子的细胞质和膜结构域之间被定义，这对其组装和功能都是至关重要的。其中一个结构域交换相互作用是由苯丙氨酸残基508的芳香侧链介导的，在大多数CF患者中缺失，我们独立发现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的多个结构域的作用以及它们之间在其正常功能中的相互作用，然后确定这些结构域如何被主要的囊性纤维化突变F508改变。第一个广泛目标将解决四个尚未解决的重大问题。第一个问题是，未水解的ATP脱离NBD2的简并特征基序及其磷酸化是否刺激了NBD1的解离，从而打开了nbd之间的界面并关闭了通道。其次，我们将确定nbd和msd之间的六个“传输接口”中的每个“传输接口”的作用，包括那些在分子的相反两侧之间介导域交换或缠绕的接口。第三，将绘制响应通道激活刺激的膜跨域螺旋间关系的变化，并确定它们对离子孔的贡献。第四，确定nbd和磷酸化控制的R结构域在CFTR功能过程中的相互影响。不同功能态的高分辨率三维结构将通过电子晶体学与这些生化研究相结合来确定。在我们将Phe508定位为3D结构的第二个主要目标中，我们将确定其缺失对蛋白质其余部分的结构和功能的影响，以促进新的治疗策略的发展。

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.