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 (Pg p), which has been implicated in the multidrug resistance of human tum ors because of its ability to act as an ATP-driven efflux pump for hyd rophobic compounds. Highly purified Pgp has been labeled on two cystei ne residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3 -benzoxadiazole). We show that NBD labels the same cysteine residues a s MIANS [2-(4-maleimidoanilino)naphthalene-6-sulfonic acid]; they are located within the Walker A motif of the nucleotide binding domain, cl ose to the site where ATP binds. NBD-and MIANS-labeled Pgps were recon stituted by detergent dilution into phospholipid vesicles containing i ncreasing mole fractions of rhodamine- or NBD-labeled phosphatidyletha nolamine (PE), respectivery. The fluorescence of the NBD-Pgp and MIANS -Pgp donors was quenched in a concentration-dependent manner by the rh odamine-PE and NBD-PE accepters. Using two different methods to analyz e Forster resonance energy transfer, the distance of the Pgp-bound pro bes from the lipid-water interfacial region of the bilayer was estimat ed to be 31-35 Angstrom. This distance is compatible with the low-reso lution structure of Pgp determined by electron microscopy, and indicat es that the nucleotide binding domains lie close to the membrane surfa ce. The experimental data fitted very well to theoretical quench curve s for a single protein-bound fluor, suggesting that the two nucleotide binding domains are located equidistant from the bilayer. Following t he addition of ATP to MIANS-Pgp, the NBD-PE quench curve no longer con formed to the models. These results imply that Pgp interacts different ly with PE when it is in the ATP-bound form.