Fig. 2.

F508del-CFTR exhibits defective phosphorylation-dependent conformational changes essential for channel gating. Top panel: In Wt-CFTR, PKA increases phosphorylation at multiple sites which include S422 in the regulatory insertion (RI) of NBD1, S660 in the regulatory extension (RE) located at the amino-terminus of the R domain and other sites in the R domain. CFTR has two adenosine triphosphate (ATP)-binding sites at the NBD dimers: site 1 is non-canonical and does not hydrolyze ATP, whereas site 2 is canonical and exhibits ATPase activity. PKA phosphorylation decreases the alpha helical content of the RE and RI which removes their steric hindrances on nucleotide-binding domain 1 (NBD1). This enhances interactions at the NBD1:NBD2 interface (necessary for ATP binding) as well as the intracellular loop 1 (ICL1):NBD1 and ICL4:NBD1 interfaces (necessary for conveying conformational changes from the NBDs to the membrane spanning domains for channel gating). Bottom panel: F508del-CFTR exhibits defective phosphorylation at S660 in the RE. F508del-CFTR may also exhibit defective phosphorylation at S422 in the RI and/or other phosphorylation sites in the R domain, but this could not be confirmed with our mass spectrometry methods (sites depicted as question marks). F508del-NBD1 retains aberrant interactions with the RE and RI upon PKA phosphorylation; this prevents NBD1 from interacting with NBD2, ICL1 and ICL4 which results in impaired ATP binding/hydrolysis and defective conformational changes necessary for channel gating