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

Tzyh-Chang Hwang,Toshihiko Ogura,Yoshiro Sohma,Hiroyasu Shimizu,Kazuhiro Mio,Chikara Sato,Muneyo Mio

doi:10.1074/jbc.m803185200

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.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR)3 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
The non-permeabilized control cells were negative to the antibody (Fig. 1a, second and fourth rows), confirming that both N and C termini are located inside the cells
Purification of CFTR Protein—The insect Sf9 expression system has been shown to express a large quantity of CFTR proteins [39]

Summary

EXPERIMENTAL PROCEDURES

Expression Constructs, Transfection of HEK293 Cells, and Membrane Preparation—Full-length human CFTR cDNA was subcloned into pcDNA3.1 Zeo (Invitrogen) and tagged with FLAG sequence at either the N (abbreviated N-FLAG CFTR) or C terminus (C-FLAG CFTR). Cells were incubated with Cy3-conjugated anti-FLAG antibodies (Sigma) in PBS containing 2 mg/ml bovine serum albumin and 0.1% saponin at 4 °C for 6 h They were washed with PBS three times and incubated with PBS containing 1 ␮g/ml of 4Ј,6-diamidino-2-phenylindole at room temperature for 30 min. Protein Purification—The membrane fraction was homogenized in 5 volumes (v/w) of buffer A (TBS containing 25 mM n-dodecyl ␤-D-maltoside (DDM) (Sigma), 300 mM MgCl2, 10% glycerol, protease inhibitors, and 0.02% sodium azide). The column was washed with 10 column volumes of buffer B (TBS containing 5 mM DDM, 300 mM MgCl2, 10% glycerol, protease inhibitors, and 0.02% sodium azide), and the bound CFTR protein was eluted with buffer B containing 100 ␮g/ml FLAG peptide (Sigma). The resolution of the final three-dimensional map was assessed using the Fourier shell correlation function [36] at the threshold of 0.5

RESULTS

To more accurately estimate the molecular mass of the purified

Structural Features of the CFTR

DISCUSSION