Phenylalanine 508 Forms Intra-domain Contact Crucial To CFTR Folding And Dynamics

Abstract

Cystic fibrosis (CF) is the most prevalent genetically inherited lethal disease in the United States, with an especially high incidence rate among people of European descent and affects at least one in every 2500-4000 newborns. In 90% of CF patients, the disease is caused by the deletion of phenylalanine-508 (Phe508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette (ABC) transporter family. Prior experimental studies suggest that the Phe508 deletion cause the aberrant folding of NBD1 and the misassembly of various CFTR domains. To gain a fuller understanding of the role of Phe508 in channel function and of the impact of its deletion, we constructed a 3D structural model of CFTR using the high-resolution structure of another ABC transporter, Sav1866. The CFTR model predicts, and we validated experimentally, that Phe508 mediates an interaction between NBD1 and the fourth cytoplasmic loop in the second membrane-spanning domain (MSD2). This interface explains the observed sensitivity of CFTR assembly to many disease-associated mutations in CL4 as well as in NBD1. We also confirmed other intra-domain contacts between the cytoplasmic and membrane-spanning domains that were predicted by the CFTR model. Furthermore, our electrophysiological measurements and molecular dynamics simulations show that these interfaces regulate channel gating and are highly dynamic. Aside from advancing understanding of CFTR structure and function, this study identifies the region of CFTR that should be targeted in treating cystic fibrosis.