The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR

Non-technical summary Cystic fibrosis is a genetic disease caused by the malfunction of a chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR). The most common disease-associated mutation is the deletion of the phenylalanine residue at position 508 (delta F508), which result in channels with poor membrane expression and defective function. Opening of CFTR channels is controlled by ATP binding at two intracellular domains, called nucleotide-binding domains (NBDs), and subsequent NBD dimerization. Our previous studies revealed that delta F508-CFTR channels open very infrequently, raising the possibility that the mutation perturbs NBD dimerization although the mutation is not located near the dimer interface. In this paper, we employed a functional assay to assess the stability of the NBD dimer. Our data suggest that the delta F508 mutation significantly destabilizes the NBD dimer, supporting the hypothesis that the mutation disrupts the dimer interface. Our results provide structural insights that are potentially useful for drug design.The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 (delta F508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N6-(2-phenylethyl)-2'-deoxy-ATP (P-dATP), can increase the open probability (P(o)) to similar to 0.7, implying that the gating defect of delta F508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PP(i)), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N6-(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PP(i)-induced locked-open time and the ATP/P-ATP ligand exchange time of delta F508-CFTR channels are dramatically shortened, suggesting that the delta F508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of delta F508-CFTR, namely the solubilizing mutation F494N/Q637R and delta RI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with delta F508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of delta F508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for delta F508 channels. The structure-function insights gained from this mechanism may provide clues for future drug design.