Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

Vinciane Grimard,Canhui Li,Mohabir Ramjeesingh,Christine E Bear,Erik Goormaghtigh,Jean-Marie Ruysschaert

doi:10.1074/jbc.m311014200

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site.

Highlights

Cystic fibrosis is caused by a mutation in the membrane chloride channel CFTR1 [1]
We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium
This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments

Summary

EXPERIMENTAL PROCEDURES

Materials—D2O was from Merck, nickel-nitrilotriacetic acid resin was from Qiagen, m3a7 CFTR primary antibody and anti-mouse secondary antibody were from Chemicon, ECLϩ Western blot detection kit was from Amersham Biosciences, pentadecafluorooctanoic acid (PFO) was from Fluorochem, SM2 Bio-Beads were from Bio-Rad, bovine cAMP-dependant protein kinase A catalytic domain was from Promega, and bovine alkaline phosphatase was from Sigma. Uptake of Chloride by Proteoliposomes—A concentrative tracer uptake assay, described previously [20], was used to measure 36ClϪ flux into proteoliposomes containing purified and reconstituted CFTR protein. A linear model containing two wavenumbers was constructed from the reference data base for each secondary structure and applied to the CFTR spectra. Both the protein data base and the mathematical model have been described previously in the case of circular dichroism [25]. Kinetics of Deuteration—Films containing 20 ␮g of reconstituted phosphorylated or dephosphorylated CFTR were prepared on a germanium plate as described above. Polarized ATR-FTIR Spectroscopy—Films containing 20 ␮g of reconstituted phosphorylated or dephosphorylated CFTR were prepared on a germanium plate as described above. Acrylamide quenching data were analyzed according to the Stern-Volmer equation for collisional quenching [30]

RESULTS

17 Ϯ 2 16 Ϯ 2

DISCUSSION