Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet.

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.