Molecular Pathophysiology of Cystic Fibrosis

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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.