Molecular Mechanisms of CFTR Function

CFTR功能的分子机制

• 批准号: 7784969
• 负责人: JOHN R RIORDAN
• 金额: $ 37万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7784969
• 关键词: ATP Hydrolysis; ATP-Binding Cassette Transporters; Address; Antibodies; Architecture; Binding; Binding Sites; Biochemical; Cell membrane; Coupled; Coupling; Cyclic AMP-Dependent Protein Kinases; Cysteine; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Detection; Development; Disease; Dissociation; Epithelial; Family; Functional disorder; Health; Homeostasis; Human; Hydrolysis; Ion Channel; Ions; Kinetics; Knowledge; Label; Ligands; Liquid substance; Maps; Marines; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; Motor; Movement; Mutate; Mutation; NBS1 gene; Nucleotides; One-Step dentin bonding system; Performance; Phenylalanine; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Positioning Attribute; Property; Proteins; Publishing; Radial; Reaction; Reagent; Regulation; Resolution; Rest; Role; Side; Signal Transduction; Site; Sodium Chloride; Stimulus; Structural Models; Structure; Surface; Testing; Vertebrates; crosslink; cystic fibrosis patients; electron crystallography; ligand gated channel; member; mutant; novel therapeutics; protein function; public health relevance; research study; response; three dimensional structure; transmission process

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.

描述（由申请人提供）：囊性纤维化跨膜电导调节剂（CFTR）在脊椎动物上皮盐和液体稳态中起着至关重要的作用，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R结构域的磷酸化状态的控制。迄今为止的发现与单体CFTR充当可水解式封闭通道的模型一致，其中磷酸化调节了ATP结合/水解和通道门口之间的变构耦合。我们最近发现，磷酸化而不是影响ATP的水解，而是促进NBD1的未溶解ATP的释放，并增加了很大程度上非结构化的R结构域的回旋半径，从而改变了膜跨度域的构象。我们是唯一通过电子晶体学纯化，结晶并确定完整蛋白质的低分辨率结构的组。我们还在计算上生成了高分辨率模型，该模型满足了一系列已发表的实验数据，并揭示了我们通过半胱氨酸交联和结合实验证实的域相互作用。在分子的相对一半的细胞质和膜结构域之间已经定义了域交换相互作用，这对其组装和功能至关重要。这些结构域交换的相互作用之一是由苯丙氨酸残基508的芳族侧链介导的，该苯丙氨酸残基508在大多数CF患者中删除，我们独立显示，这直接参与了通道门控。我们现在的主要目标是进一步阐明野生型CFTR多个域的作用以及它们在其正常功能中的相互作用，然后确定它们如何被主要的囊性纤维化导致突变的主要囊性纤维化改变。第一个广泛的目标将解决四个重要的未解决问题。第一个询问NBD2的退化信号基序及其磷酸化是否刺激了NBD1的解离，这是否有助于NBDS和通道关闭之间的界面开放。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，包括介导分子相对侧之间的域交换或交织的六个“传输接口”。第三，将映射膜间跨度域中螺旋之间关系的变化，并将绘制对通道激活刺激的响应，并将其对所鉴定的离子孔的贡献。第四，将确定NBD的影响和磷酸化控制的R结构域在CFTR函数期间相互互相控制。不同功能状态的较高分辨率3D结构将由电子晶体学结合这些生化研究确定。在我们将PHE508定位在3D结构中的第二个主要目标中，我们将确定其缺失对蛋白质其余蛋白质的结构和功能的影响，以促进新的治疗策略的发展。 公共卫生相关性：CFTR是一种独特的离子通道，采用改良的活性转运蛋白结构结构，当突变时会导致人类的囊性纤维化。磷酸化调节的通道在几乎所有陆生和海洋脊椎动物的上皮表面上提供了离子和流体运动的速率限制步骤。因此，对于理解正常的人类健康和疾病，对其3D结构和动态的了解对于理解正常的人类健康和疾病至关重要。