title: Regulatory insertion removal restores maturation, stability and function of DeltaF508 CFTR.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multidomain membrane protein that matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide-binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32-residue segment termed the regulatory insertion (RI) not present in other ATP-binding cassette transporters. We report here that RI deletion enabled F508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long-term pulse-chase experiments showed that the mature DeltaRI/DeltaF508 had a T(1/2) of approximately 14 h in cells, similar to the wild type. RI deletion restored ATP occlusion by NBD1 of DeltaF508 CFTR and had a strong thermostabilizing influence on the channel with gating up to at least 40 degrees C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508-containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
authors.affiliation: Department of Biomedical Engineering, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Aleksandrov
authors.forename: Pradeep
authors.initials: P
authors.lastname: Kota
authors.forename: Luba A
authors.initials: LA
authors.lastname: Aleksandrov
authors.forename: Lihua
authors.initials: L
authors.lastname: He
authors.forename: Tim
authors.initials: T
authors.lastname: Jensen
authors.forename: Liying
authors.initials: L
authors.lastname: Cui
authors.forename: Martina
authors.initials: M
authors.lastname: Gentzsch
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: C505032:CFTR protein, human; D050505:Mutant Proteins; D011994:Recombinant Proteins; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate