NMR evidence for differential phosphorylation-dependent interactions in WT and DeltaF508 CFTR.
The most common cystic fibrosis (CF)-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of Phe508 (DeltaF508) in the first of two nucleotide-binding domains (NBDs). Nucleotide binding and hydrolysis at the NBDs and phosphorylation of the regulatory (R) region are required for gating of CFTR chloride channel activity. We report NMR studies of wild-type and DeltaF508 murine CFTR NBD1 with the C-terminal regulatory extension (RE), which contains residues of the R region. Interactions of the wild-type NBD1 core with the phosphoregulatory regions, the regulatory insertion (RI) and RE, are disrupted upon phosphorylation, exposing a potential binding site for the first coupling helix of the N-terminal intracellular domain (ICD). Phosphorylation of DeltaF508 NBD1 does not as effectively disrupt interactions with the phosphoregulatory regions, which, along with other structural differences, leads to decreased binding of the first coupling helix. These results provide a structural basis by which phosphorylation of CFTR may affect the channel gating of full-length CFTR and expand our understanding of the molecular basis of the DeltaF508 defect.
Kanelis
Voula
V
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada.
Hudson
Rhea P
RP
Thibodeau
Patrick H
PH
Thomas
Philip J
PJ
Forman-Kay
Julie D
JD
D000818:Animals; D001665:Binding Sites; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D011994:Recombinant Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator