Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: a resonance energy transfer study.
Very little structural information is available for P-glycoprotein (Pgp), which has been implicated in the multidrug resistance of human tumors because of its ability to act as an ATP-driven efflux pump for hydrophobic compounds. Highly purified Pgp has been labeled on two cysteine residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). We show that NBD labels the same cysteine residues as MIANS [2-(4-maleimidoanilino)naphthalene-6-sulfonic acid]; they are located within the Walker A motif of the nucleotide binding domain, close to the site where ATP binds. NBD- and MIANS-labeled Pgps were reconstituted by detergent dilution into phospholipid vesicles containing increasing mole fractions of rhodamine- or NBD-labeled phosphatidylethanolamine (PE), respectively. The fluorescence of the NBD-Pgp and MIANS-Pgp donors was quenched in a concentration-dependent manner by the rhodamine-PE and NBD-PE acceptors. Using two different methods to analyze Förster resonance energy transfer, the distance of the Pgp-bound probes from the lipid-water interfacial region of the bilayer was estimated to be 31-35 A. This distance is compatible with the low-resolution structure of Pgp determined by electron microscopy, and indicates that the nucleotide binding domains lie close to the membrane surface. The experimental data fitted very well to theoretical quench curves for a single protein-bound fluor, suggesting that the two nucleotide binding domains are located equidistant from the bilayer. Following the addition of ATP to MIANS-Pgp, the NBD-PE quench curve no longer conformed to the models. These results imply that Pgp interacts differently with PE when it is in the ATP-bound form.
Liu
R
R
Guelph-Waterloo Centre for Graduate Work in Chemistry and Biochemistry, Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
Sharom
F J
FJ
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane; D004735:Energy Transfer; D005454:Fluorescence Polarization; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008956:Models, Chemical; D013329:Structure-Activity Relationship
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007202:Indicators and Reagents; D008563:Membrane Lipids; D000255:Adenosine Triphosphate; D009327:4-Chloro-7-nitrobenzofurazan