Molecular Pathophysiology of Cystic Fibrosis

cftr基因的功能丧失突变是囊性纤维化（CF）的根本原因， 美国第二常见的缩短寿命的遗传疾病。作为ABC的一员， (ATP结合盒）转运蛋白超家族，CFTR蛋白包含两个 跨膜结构域（TMD 1和TMD 2），每个后接核苷酸结合区， 由典型的步行者A和B表征的结构域（分别为NBD 1和NBD 2 用于ATP结合/水解的基序，和特征序列（即，LSGGQ），它扮演一个 在ATP结合后，NBD二聚体的形成起关键作用。CFTR是 独特之处在于，CFTR不是一种活性转运蛋白，而是一种真正的离子通道。 我们以前的研究已经导致了CFTR的门控机制，其特征在于 ATP诱导的NBD二聚化和门开放之间的概率关系 TMD CFTR的三种低温电磁结构的最新解决方案，一方面，争议 我们的想法是，通道关闭不需要完全分离两个NBD，但 另一方面，支持我们的主张，即NBD二聚化并不能保证门 开放后这些低温EM数据缺乏高的时间分辨率，但其精致的空间 决议确实为我们提供了前所未有的机会来解决几个悬而未决的问题 关于CFTR功能解剖的问题（目标1），例如： 冷冻EM结构中完全分离的NBD的功能意义？ 两个ATP结合位点如何影响彼此的功能？一个封闭的存在 具有二聚NBD的通道不仅需要更深入的门控研究， 机制，但也迫使我们挑战长期持有的观点，即只有开放的 通道水解ATP。由于许多疾病相关的突变存在于NBD中， 二聚体界面，因此是很好的主题，促进我们的调查， NBD/TMD耦合，所提出的研究还可以揭示NBD/TMD耦合的机制， 这些突变在分子水平上引起CF。我们的一个重要应用 CFTR门控的基础研究是利用知识来探索药物或 候选药物影响CFTR门控的不同方面（目标2），并确定是否和什么 致病性突变对治疗试剂的反应程度（目的3）。与原子 CFTR在不同状态下的结构，及时探索试剂如何通过结合不同的 CFTR区域协同激活CFTR。讨论后一个问题可以作为一个 实现基于结构的药物设计的垫脚石。

Loss-of-function mutations in the cftr gene are the root cause of cystic fibrosis (CF), the second most common life-shortening genetic disease in the US. As a member of the ABC (ATP Binding Cassette) transporter superfamily, the CFTR protein comprises two transmembrane domains (TMD1 and TMD2), each followed by a nucleotide binding domain (NBD1 and NBD2 respectively) characterized by the canonical Walker A and B motifs for ATP binding/hydrolysis, and a signature sequence (i.e., LSGGQ) that plays a critical role for the formation of a head-to-tail NBD dimer upon ATP binding. CFTR is unique in that, instead of being an active transporter, CFTR is a bona fide ion channel. Our previous studies have led to a gating mechanism of CFTR that features a probabilistic relationship between ATP-induced NBD dimerization and gate opening in the TMDs. Recent solutions of three cryo-EM structures of CFTR, on one hand, dispute our idea that channel closure does not require a complete separation of the two NBDs, but on the other hand, support our proposition that NBD dimerization does not guarantee gate opening. These cryo-EM data lack high temporal resolution, but their exquisite spatial resolution does offer us an unprecedented opportunity to address several unsettled questions regarding the functional anatomy of CFTR (Aim 1) such as: What is the functional significance of completely separated NBDs shown in the cryo-EM structures? How do the two ATP-binding sites affect each other’s function? The existence of a closed channel with dimerized NBDs not only demands more thorough studies of the gating mechanism but also compels us to challenge the long-held view that only the open channel hydrolyzes ATP. As many of the disease-associated mutations reside in the NBD dimer interface, and thus are excellent subjects facilitating our investigation into NBD/TMD coupling, the proposed studies could also reveal the mechanism by which these mutations cause CF at a molecular level. One important application of our fundamental studies of CFTR gating is to use the knowledge to explore how drugs or drug candidates affect different aspects of CFTR gating (Aim 2), and to determine if and to what extent pathogenic mutations respond to therapeutic reagents (Aim 3). Together with the atomic structures of CFTR in different states, it is timely to probe how reagents, by binding to different regions of CFTR, synergistically activate CFTR. Answering this latter question could serve as a stepping-stone to materialize structure-based drug design.