Molecular Mechanisms of CFTR Function

CFTR功能的分子机制

• 批准号: 8233336
• 负责人: JOHN R RIORDAN
• 金额: $ 33.33万
• 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/8233336
• 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器（CFTR）在脊椎动物上皮盐和液体稳态中发挥着关键作用，其缺失或功能障碍会导致人类囊性纤维化。在这个项目中，我们描述了 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 结构域磷酸化状态的控制。迄今为止的研究结果与单体 CFTR 作为可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耦合。我们最近发现，磷酸化不是影响 ATP 水解，而是促进 NBD1 释放未水解的 ATP，并且还增加了很大程度上非结构化的 R 结构域的回转半径，从而改变了跨膜结构域的构象。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域相互作用。分子相对半部分的细胞质和膜结构域之间的结构域交换相互作用已被定义，这对其组装和功能至关重要。这些结构域交换相互作用之一是由苯丙氨酸残基 508 的芳香族侧链介导的，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 多个结构域的作用以及它们在其正常功能中的相互作用，然后确定这些结构如何被导致囊性纤维化的主要突变 ?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 与 NBD2 简并特征基序的脱离及其磷酸化刺激的与 NBD1 的解离是否有助于 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“传输界面”中每一个的作用，包括那些介导分子相对侧之间的结构域交换或交织的界面。第三，将绘制跨膜域中螺旋间关系响应通道激活刺激的变化，并确定它们对离子孔的贡献。第四，将确定在 CFTR 功能期间 NBD 和磷酸化控制的 R 结构域相互之间的影响。不同功能状态的更高分辨率 3D 结构将通过电子晶体学结合这些生化研究来确定。在我们将 Phe508 在 3D 结构中定位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，以促进新治疗策略的开发。 公共健康相关性：CFTR 是一种独特的离子通道，采用改良的主动转运蛋白结构架构，突变时会导致人类囊性纤维化。在几乎所有陆地和海洋脊椎动物中，磷酸化调节通道为离子和液体跨上皮表面的运动提供了速率限制步骤。因此，了解其在执行此功能期间的 3D 结构和动力学对于理解正常的人类健康和疾病具有基础和实际的重要性。