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Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface.

J Biol Chem. 2013 Jun 3;

Authors: Loo TW, Bartlett MC, Clarke DM


The P-glycoprotein (P-gp) drug pump protects us from toxins. Drug-binding sites in the transmembrane (TM) domains (TMDs) are connected to the nucleotide-binding domains (NBDs) by intracellular helices (IHs). TMD-NBD crosstalk is a key step in the transport mechanism since drug binding stimulates ATP hydrolysis followed by drug efflux. Here, we tested whether the IHs are critical for maturation and TMD-NBD coupling by characterizing the effects of mutations to the IH1 and IH2 interfaces. While IH1 mutations had little effect, most mutations at the IH2-NBD2 interface inhibited maturation or activity. For example, the F1086A mutation at the IH2-NBD2 interface abolished drug-stimulated ATPase activity. The mutant F1086A however, retained the ability to bind ATP and drug substrates. The mutant was defective in mediating ATP-dependent conformational changes in the TMDs because binding of ATP no longer promoted cross-linking between cysteines located at the extracellular ends of TM segments 6 and 12. Replacement of Phe1086 (in NBD2) with hydrophobic but not charged residues yielded active mutants. The activity of the F1086A mutant could be restored when the nearby residue Ala266 (in IH2) was replaced with aromatic residues. These results suggest that Phe1086/Ala266 lies in a hydrophobic IH2-NBD2 ball-and-socket joint.

PMID: 23733192 [PubMed - as supplied by publisher]