Domain location within the cystic fibrosis transmembrane conductance regulator protein investigated by electron microscopy and gold labelling.
The domain organisation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein was studied using electron microscopy of detergent-solubilised dimeric complexes. Ni-NTA nanogold labelling data suggest that in the nonphosphorylated, nucleotide-free state, the C-terminus is intimately associated with the cytoplasmic ATP-binding regions, whilst part of the regulatory domain occupies a position close to the cytoplasmic surface of the lipid membrane. Removal of the entire second nucleotide binding domain (NBD2) results in a deficit in the CFTR structure that is consistent with the size and shape of a single NBD. The data suggest that NBD2 lies closer to the C2 symmetry axis than the first nucleotide binding domain (NBD1) and that NBD2 from one CFTR monomer also contacts NBD1 from the opposing one. These data suggest that current homology models for CFTR based on other ATP-binding cassette proteins appear to be reasonable, at least to low resolution. We also find that Ni-NTA nanogold labelling of an internal hexa-Histidine sequence is a valuable approach to locate individual protein domains.
Zhang
Liang
L
Faculty of Life Sciences, The University of Manchester, MIB, 131 Princess St., Manchester M1 7DN, UK.
Aleksandrov
Luba A
LA
Riordan
John R
JR
Ford
Robert C
RC
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008055:Lipids / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods; D036103:Nanotechnology / Q000379:methods; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
C505032:CFTR protein, human; D008055:Lipids; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold; D000255:Adenosine Triphosphate