Three-dimensional Reconstruction of Human Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Revealed an Ellipsoidal Structure with Orifices beneath the Putative Transmembrane Domain

• Published in: The Journal of biological chemistry Vol. 283; no. 44; pp. 30300 - 30310
• Main Authors: Mio, Kazuhiro, Ogura, Toshihiko, Mio, Muneyo, Shimizu, Hiroyasu, Hwang, Tzyh-Chang, Sato, Chikara, Sohma, Yoshiro
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.10.2008; American Society for Biochemistry and Molecular Biology
• Subjects: Cell Line; Cell Membrane - metabolism; Chromatography - methods; Cross-Linking Reagents - pharmacology; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Cytoplasm - metabolism; DNA, Complementary - metabolism; Gene Expression Regulation; Humans; Image Processing, Computer-Assisted; Kinetics; Microscopy, Electron, Transmission; Protein Binding; Protein Conformation; Protein Structure and Folding; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M803185200

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 × 106 × 162Å. 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.