The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding
cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common
mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated
mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops
(CL) of the membrane-spanning domains (MSD). A recently constructed computational
model of the CFTR three-dimensional structure, supported by experimental data (Serohijos,
A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan,
J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several
of these mutations including DeltaF508 disrupted interfaces between these domains.
Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces
predicted by the structural model and observed that their cross-linking has a variety
of different effects on channel gating. The interdomain contacts comprise aromatic
clusters important for stabilization of the interfaces and also involve the Q-loops
and X-loops that are in close proximity to the ATP binding sites. Cross-linking of
all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves
of the protein rapidly and reversibly arrest single channel gating while those in
the same halves have lesser impact. These results reinforce the idea that mediation
of regulatory signals between cytoplasmic- and membrane-integrated domains of the
CFTR channel apparently relies on an array of precise but highly dynamic interdomain
structural joints.
