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 ؋ 106 ؋ 162 Å . 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 CFTR⅐anti-regulatory domain antibody complex confirmed that two regulatory domains are located around the bottom end of the larger oval cytoplasmic domain.The cystic fibrosis transmembrane conductance regulator (CFTR) 3 (also termed ABCC7) is a unique member of the ATPbinding cassette (ABC) superfamily in that CFTR functions as an anion channel, whereas most other members function as active transporters. CFTR is expressed in the luminal membranes of secreting and absorbing epithelia and plays a critical role in transepithelial salt and water transport. Dysfunction of CFTR leads to cystic fibrosis, the most common lethal autosomal recessive disorder in Caucasians (1-3). On the other hand, extremely high activity of CFTR, usually caused by bacterial toxins, results in secretory diarrhea (4, 5), killing millions of infants in developing countries every year (6). Understanding the structure/function relationship and the underlying mechanisms of CFTR are essential for developing novel therapeutics for CFTR-mediated diseases.Like other ABC transporters, CFTR is formed by two repeated motifs, each of which has a membrane-spanning domain (MSD) and a cytoplasmic nucleotide-binding domain (NBD). However, a regulatory domain (R domain) located between the first NBD (NBD1) and the second MSD (MSD2) is unique in CFTR among ABC transporters. This domain contains several phosphorylation sites for protein kinase A and protein kinase C, and the level of their phosphorylation controls CFTR channel activity. Once they are phosphorylated, opening and closing (gating) of the CFTR channel is controlled by ATP binding and hydrolysis at its two NBDs (7). Each NBD contains the Walker A and Walker B nucleotidebinding motifs and the "signature sequence" LSGGQ, which defines the...

Molecular Mechanisms of TRPV1 Channel Activation

“…A recently obtained tridimensional reconstruction of the channel at 19 Å resolution by cryo-electronmycroscopy [79] indicated that it is similar, in terms of size and topology of both the transmembrane-and intracellular regions, to the mammalian Kv1.2 channel, whose structure has been determined at high resolution [80]. Similar studies with TRPM2 [81] and TRPC3 [82] suggest that these channels have more dilated intracellular structures as compared to Kv channels, and are more similar to the cystic fibrosis transmembrane regulator of conductance, CFTR [83]. In the case of TRPC3, these differences might arise from accessory subunits associated with the channel or from lipid aggregates included in the analysis [82,84].…”

Molecular models of the open and closed states of the whole human CFTR protein

“…Recently, a first clear 3D structural reconstruction of CFTR obtained at 20 Å resolution by electron microscopy (EM) was reported [28], offering an experimental validation tool for modeling studies. This work supported a ''tail-to-tail'' dimeric assembly of the complete mature CFTR molecule (169 kDa ?…”

“…Furthermore, we also tentatively proposed a two-domain model of the regulatory (R) domain and indicated how this model might accommodate the two states of CFTR. Finally, we also evaluated how our present model of the overall CFTR MSD1:NBD1:R:MSD2:NBD2 assembly fits with recent structural data obtained by electron microscopy [28] and cryo-electron microscopy [29], as well as with previous work with electron crystallography [30].…”

Tetrameric Orai1 Is a Teardrop-shaped Molecule with a Long, Tapered Cytoplasmic Domain

“…Estimation of Stokes Radius by SEC-High molecular weight standard proteins (GE Healthcare) and purified Orai1 protein were analyzed by Superdex 200 (3.2/30) SEC in a Smartsystem (35). The elution profile of each protein was obtained at 280 nm.…”