DEVELOPMENT OF MEMBRANE PROTEIN STRUCTURE

描述(由申请人提供)：囊性纤维化传导调节蛋白(CFTR)是ABC转运体超基因家族的成员之一，是一种多基因整合的膜蛋白。这些蛋白质由两个胞质核苷酸结合域(NBD)和两个跨膜结构域(TMD)组成，它们折叠并结合形成功能性的ABC酶，利用ATP结合和水解来介导跨膜事件。与其他ABC转运蛋白不同的是，CFTR还包含一个独特的、额外的、基本上无序的部分，称为R区。编码cftr的基因突变会导致囊性纤维化，通常是通过扰乱蛋白质的折叠和组装进入其功能天然结构。错误折叠的突变蛋白被细胞中针对其降解的质量控制蛋白识别。这些突变中最常见的是？F508，它位于NBD1表面的一个区域，该区域与其他ABC转运蛋白中的TMD相互作用。因此，关于？F508突变的分子病理学的三个基本问题出现了。突变会破坏NBD1的折叠吗？突变是否会破坏NBD1与CFTR或其伙伴蛋白的其他结构域之间的关联？如何识别未有效折叠的CFTR？承诺的识别步骤是什么？提出了三个具体的目标来解决这些问题，并进一步提供了对多面体膜蛋白折叠更一般过程的洞察。它们是：确定？F508突变对NBD1折叠和动力学的影响。野生型和？F508NBD1的天然状态非常相似，但突变体更容易形成部分折叠状态。这种状态的结构和动力学将被描述出来。其他与F508相互作用影响这些状态之间转换的残基将使用计算和遗传方法来确定。阐明？F508突变对NBD1与CFTR其他结构域相互作用的影响。最近的晶体结构表明，508侧链暴露在NBD1表面靠近预测的界面附近，细胞内环(ICL)连接CFTR的跨膜跨膜。使用细胞生物学、生化、结构和计算方法，将鉴定与NBD1相互作用的ICL，并确定？F508突变对相互作用强度的影响。还将利用现有的方法来监测与R区和其他结构域的相互作用。确定在折叠的最早步骤与新生的CFTR链相互作用的蛋白质，以及当折叠被致病突变(如？F508)破坏时相互作用的蛋白质。一种强大的生物合成方法，将特定部位的光亲和探针结合到已定义的CFTR新生链中，将被用于鉴定在折叠和整合的最早步骤中相互作用的蛋白质。特别令人感兴趣的是蛋白质的同一性，初步数据表明，优先识别具有特定CF引起突变的新生链条。这些研究对于从根本上理解膜蛋白结构的发展和规避CF分子病理的策略是必要的。与公共卫生相关。囊性纤维化是一种常见的致命遗传病，大多数病例是由干扰囊性纤维化跨膜传导调节蛋白(CFTR)组装的突变引起的。拟议的研究将阐明致病突变如何干扰这一过程的细节。了解组装过程，从而了解详细的分子病理学，将为开发囊性纤维化的靶向治疗提供重要信息。

DESCRIPTION (provided by applicant): The cystic fibrosis conductance regulator (CFTR) is a polytopic integral membrane protein that is a member of the ABC transporter supergene family. These proteins are composed of two cytosolic nucleotide binding domains (NBDs) and two transmembrane domains (TMDs) that fold and associate to form the functional ABC enzymes which utilize ATP binding and hydrolysis to mediate transmembrane events. In contrast to other ABC transporters, CFTR also contains a unique, additional, largely-disordered portion called the R region. Mutations in the gene that encodes CFTR cause cystic fibrosis, typically by disrupting the folding and assembly of the protein into its functional native structure. The misfolded mutant protein is recognized by quality control proteins in the cell that target it for degradation. The most common of these mutations is ?F508 which is found on the surface of NBD1 in a region that mediates interactions with the TMDs in other ABC transporters. Thus, three fundamental questions about the molecular pathology of the ?F508 mutation arise. Does the mutation disrupt the folding of the NBD1? Does the mutation disrupt the association of NBD1 and other domains of CFTR or its partner proteins? How is CFTR that is not efficiently folded identified and what is the committed recognition step? Three specific aims are proposed to address these questions and, further, to provide insight into the more general process of polytopic membrane protein folding. They are: Define the effects of the ?F508 mutation on the folding and dynamics of NBD1. The native states of wild type and ?F508 NBD1 are remarkably similar, but the mutant more readily populates a partially folded state. The structure and dynamics of this state will be characterized. Other residues that interact with F508 to affect conversions between these states will be identified using computational and genetic approaches. Elucidate the effects of the ?F508 mutation on the interactions of NBD1 with other domains of CFTR. Recent crystal structures indicate that the 508 side chain is exposed on the surface of NBD1 near a predicted interface with intracellular loops (ICLs) connecting the transmembrane spans of CFTR. Using cell biological, biochemical, structural, and computational methods, the ICLs that interact with NBD1 will be identified and the effect of the ?F508 mutation on the strength of the interaction determined. Extant methods for monitoring interactions with the R region and other domains will also be utilized. Identify the proteins that interact with the nascent CFTR chain at the earliest steps of folding and those that interact when folding is disrupted by disease-causing mutations such as ?F508. A powerful biosynthetic method of incorporating site-specific photoaffinity probes in defined CFTR nascent chains will be used to identify proteins that interact during the earliest steps of folding and integration. Of particular interest are the identities of the proteins preliminary data indicate preferentially recognize nascent chains with specific CF-causing mutations. These studies are necessary for a fundamental understanding of the development of membrane protein structure and strategies to circumvent the molecular pathology of CF. PUBLIC HEALTH RELEVANCE. Most cases of cystic fibrosis, a common fatal genetic disease, are caused by mutations that interfere with the assembly of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The studies proposed will elucidate the details of how the disease-causing mutations interfere with this process. Understanding the assembly process, and, thus, the detailed molecular pathology, will provide important information for developing targeted therapeutics for cystic fibrosis.