title: Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a membrane-integral protein that belongs to an ATP-binding cassette superfamily. Mutations in the CFTR gene cause cystic fibrosis in which salt, water, and protein transports are defective in various tissues. Here we expressed wild-type human CFTR as a FLAG-fused protein in HEK293 cells heterologously and purified it in three steps: anti-FLAG and wheat germ agglutinin affinity chromatographies and size exclusion chromatography. The stoichiometry of the protein was analyzed using various biochemical approaches, including chemical cross-linking, blue-native PAGE, size exclusion chromatography, and electron microscopy (EM) observation of antibody-decorated CFTR. All these data support a dimeric assembly of CFTR. Using 5,039 automatically selected particles from negatively stained EM images, the three-dimensional structure of CFTR was reconstructed at 2-nm resolution assuming a 2-fold symmetry. CFTR, presumably in a closed state, was shown to be an ellipsoidal particle with dimensions of 120 x 106 x 162 A. It comprises a small dome-shaped extracellular and membrane-spanning domain and a large cytoplasmic domain with orifices beneath the putative transmembrane domain. EM observation of CFTR.anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.
authors: Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan.
authors: Kazuhiro
authors: K
authors: Mio
authors: Toshihiko
authors: T
authors: Ogura
authors: Muneyo
authors: M
authors: Mio
authors: Hiroyasu
authors: H
authors: Shimizu
authors: Tzyh-Chang
authors: TC
authors: Hwang
authors: Chikara
authors: C
authors: Sato
authors: Yoshiro
authors: Y
authors: Sohma
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D019005:Cystic Fibrosis Transmembrane Conductance Regulator