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The coupling mechanism of P ‐glycoprotein involves residue L339 in the sixth membrane spanning segment
Author(s) -
Rothnie Alice,
Storm Janet,
McMahon Roisin,
Taylor Andrew,
Kerr Ian D.,
Callaghan Richard
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.06.030
Subject(s) - chemistry , residue (chemistry) , biochemistry , transmembrane protein , atp hydrolysis , binding site , transmembrane domain , affinity label , plasma protein binding , biophysics , conformational change , nucleotide , stereochemistry , enzyme , membrane , biology , receptor , atpase , gene
The transmembrane (TM) domains in P ‐glycoprotein ( P ‐gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site‐directed labelling approach was used to attach thiol‐reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin‐maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P ‐gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P ‐gp with [ 3 H]‐azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.