Can two wrongs make a right? F508del-CFTR ion channel rescue by second-site mutations in its transmembrane domains

Deletion of phenylalanine 508 (F508del), in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, is the most common cause of cystic fibrosis (CF). F508 is located on nucleotide-binding domain 1 (NBD1) in contact with cytosolic extensions of transmembrane helices, in particular intracellular loop 4 (ICL4). We carried out a mutagenesis scan of ICL4 by introducing five or six second-site mutations at eleven positions in cis with F508del, and quantifying changes in membrane proximity and ion-channel function of CFTR. The scan strongly validated the effectiveness of R1070W at rescuing F508del defects. Molecular dynamics simulations highlighted two features characterizing the ICL4/NBD1 interface of F508del/R1070W-CFTR: flexibility, with frequent transient formation of interdomain hydrogen bonds, and loosely stacked aromatic sidechains, (F1068, R1070W, and F1074, mimicking F1068, F508 and F1074 in wild-type CFTR). F508del-CFTR had a distorted aromatic stack, with F1068 displaced towards space vacated by F508. In F508del/R1070F-CFTR, which largely retained F508del defects, R1070F could not form hydrogen bonds, and the interface was less flexible. Other ICL4 second-site mutations which partially rescued F508del-CFTR are F1068M and F1074M. Methionine side chains allow hydrophobic interactions without the steric rigidity of aromatic rings, possibly conferring flexibility to accommodate the absence of F508 and retain a dynamic interface. Finally, two mutations identified in a yeast scan (A141S and R1097T, on adjacent transmembrane helices linked to ICL1 and ICL4) also partially rescued F508del-CFTR function. These studies highlight the importance of hydrophobic interactions and conformational flexibility at the ICL4/NBD1 interface, advancing understanding of the structural underpinning of F508del dysfunction.