title: Corrector VX-809 promotes interactions between cytoplasmic loop one and the first nucleotide-binding domain of CFTR.
abstract: A large number of correctors have been identified that can partially repair defects in folding, stability and trafficking of CFTR processing mutants that cause cystic fibrosis (CF). The best corrector, VX-809 (Lumacaftor), has shown some promise when used in combination with a potentiator (Ivacaftor). Understanding the mechanism of VX-809 is essential for development of better correctors. Here, we tested our prediction that VX-809 repairs folding and processing defects of CFTR by promoting interactions between the first cytoplasmic loop (CL1) of transmembrane domain 1 (TMD1) and the first nucleotide-binding domain (NBD1). To investigate whether VX-809 promoted CL1/NBD1 interactions, we performed cysteine mutagenesis and disulfide cross-linking analysis of Cys-less TMD1 (residues 1-436) and ΔTMD1 (residues 437-1480; NBD1-R-TMD2-NBD2) truncation mutants. It was found that VX-809, but not bithiazole correctors, promoted maturation (exited endoplasmic reticulum for addition of complex carbohydrate in the Golgi) of the ΔTMD1 truncation mutant only when it was co-expressed in the presence of TMD1. Expression in the presence of VX-809 also promoted cross-linking between R170C (in CL1 of TMD1 protein) and L475C (in NBD1 of the ΔTMD1 truncation protein). Expression of the ΔTMD1 truncation mutant in the presence of TMD1 and VX-809 also increased the half-life of the mature protein in cells. The results suggest that the mechanism by which VX-809 promotes maturation and stability of CFTR is by promoting CL1/NBD1 interactions.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Tip W
authors.initials: TW
authors.lastname: Loo
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Electronic address: david.clarke@utoronto.ca.
authors.forename: David M
authors.initials: DM
authors.lastname: Clarke
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D000631:Aminopyridines / Q000378:metabolism*; D052117:Benzodioxoles / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding / Q000502:physiology
chemical_list: D000631:Aminopyridines; D052117:Benzodioxoles; C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C569105:lumacaftor
keywords: 15Jf (PubChem CID: 11958611); Bithiazole correctors; CFTR; Cycloheximide (PubChem CID: 6197); Cysteine cross-linking; Cystic fibrosis; Cytoplasmic loop; Transmembrane- and nucleotide-binding domains; VX-770 (Ivacaftor, Kalydeco) (PubChem CID: 16220172); VX-809 (Lumacaftor); VX-809 (Lumacaftor) (PubChem CID: 16678941)