Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection-Fourier transform IR spectroscopy and fluorescence spectroscopy.
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.
Grimard
Vinciane
V
Center for Structural Biology and Bioinformatics, Free University of Brussels, Campus Plaine CP206/2, 1050 Brussels, Belgium.
Li
Canhui
C
Ramjeesingh
Mohabir
M
Bear
Christine E
CE
Goormaghtigh
Erik
E
Ruysschaert
Jean-Marie
JM
D000818:Animals; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007313:Insecta; D007477:Ions; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016622:Silver Staining; D013050:Spectrometry, Fluorescence / Q000379:methods; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013997:Time Factors
C505032:CFTR protein, human; D002712:Chlorides; D007477:Ions; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases