Optimization of the NBD1 Site Improves the Function of G551D-CFTR Channels

Abstract

CFTR chloride channels comprise two nucleotide binding domains (NBD1 and NBD2). Mutations (for example, G551D) that impair CFTR function result in the lethal genetic disease cystic fibrosis (CF). It's established that the opening and closing of CFTR are mainly controlled by ATP binding and hydrolysis respectively in NBD2 while ATP binding at NBD1 can modulate the stability of the open state. Using a non-hydrolytic ligand MgPPi, we locked open CFTR channels with mutations at NBD1. We found that two W401 mutations significantly increased the lock-open time (W401F: 72±3s; W401Y: 51±6s; WT: 27±2s). These gain-of-function mutations are unexpected since the W401-equivalent residue Y1219 in NBD2 can be effectively replaced by tryptophan but not phenylalanine. As the ATP molecule bound in NBD1 may interact with NBD2's signature sequence (LSHGH), we extended our study to this region. We found that reverting the histidine residue 1348 to the canonical glycine (H1348G) similarly stabilized the lock-open state (τ=67±8s). Once W401F, H1348G, or W401F/H1348G mutations were incorporated into G551D channels which no longer respond to ATP, the mutant channels become ATP-responsive with basal activity increased by ∼10-fold for W401F/G551D-, ∼4-fold for H1348G/5G551D-, and ∼25-fold for W401F/H1348G/G551D-channels. The increased channel activity is mainly due to an increased open-time. The results shown here suggest that NBD1 and NBD2 may employee different chemical mechanisms in binding ATP and that NBD1 can be a potential molecular target for developing CFTR potentiators for CF-related mutants. The effects of different nucleotides (for example, GTP and UTP) on NBD1 and NBD2 will be studied to gain a better understanding of the chemical mechanism underlying nucleotide-NBD interaction.