1. The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment
- Author
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Róisín M. McMahon, Ian D. Kerr, Janet Storm, Richard Callaghan, Andrew M Taylor, and Alice Rothnie
- Subjects
Insecta ,Time Factors ,Paclitaxel ,Protein Conformation ,Biophysics ,Plasma protein binding ,P-glycoprotein ,Multidrug resistance ,Vinblastine ,Biochemistry ,Catalysis ,Cell Line ,Residue (chemistry) ,Nicardipine ,Coupling ,Protein structure ,Adenosine Triphosphate ,Structural Biology ,ATP hydrolysis ,Coumarins ,Genetics ,Animals ,Protein Isoforms ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Cysteine ,Sulfhydryl Compounds ,Binding site ,Molecular Biology ,Adenosine Triphosphatases ,Binding Sites ,Chemistry ,Hydrolysis ,Cell Membrane ,Drug transport ,Cell Biology ,Transmembrane protein ,Recombinant Proteins ,Protein Structure, Tertiary ,Transmembrane domain ,Kinetics ,Protein Transport ,Cyclic nucleotide-binding domain ,Linear Models ,Mutagenesis, Site-Directed ,Quinolines ,Baculoviridae ,Protein Binding - Abstract
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 [3H]-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.
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