Three-dimensional reconstruction of human cystic fibrosis transmembrane conductance regulator chloride channel revealed an ellipsoidal structure with orifices beneath the putative transmembrane domain.
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.
Mio
Kazuhiro
K
Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology, Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan.
Ogura
Toshihiko
T
Mio
Muneyo
M
Shimizu
Hiroyasu
H
Hwang
Tzyh-Chang
TC
Sato
Chikara
C
Sohma
Yoshiro
Y
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
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D019005:Cystic Fibrosis Transmembrane Conductance Regulator