Ligand-dependent changes in accessibility of purified P-glycoprotein, functionally reconstituted in liposomes, were investigated by fluorescence measurements. Trp quenching experiments provided evidence that P-glycoprotein adopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATP␥S. Five anthracycline derivatives were tested as drug substrates: daunorubicin, 4-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and methoxy-morpholino-doxorubicin. Among them, daunorubicin and 4-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, whereas the three others have been shown to accumulate in multidrug-resistant cells overexpressing P-glycoprotein and therefore retain their cytotoxic activity. A small conformational change was associated with nucleotide binding and amplified after nucleotide hydrolysis. Different conformational states were adopted by P-glycoprotein upon the addition of the anthracycline derivatives in the absence and presence of MgATP or MgATP␥S. These conformational changes are shown to be related to the nature of the antitumor agents and more precisely to their capacity to accumulate in resistant cells. These data also suggest that the cytotoxicity of iododoxorubicin and 4-demethoxy-daunorubicin is related to the fact they are not transported by P-glycoprotein. On the contrary, methoxymorpholino-doxorubicin cytotoxicity may be explained in terms of its rapid reincorporation into the plasma membrane after being transported by P-glycoprotein.P-glycoprotein is a 170-kDa plasma membrane protein involved in the multidrug resistance phenomenon responsible for failure of many human cancer chemotherapies (1). A structural prediction based on its sequence predicts two homologous halves, each containing six putative membrane-spanning ␣ helices and a cytoplasmic nucleotide-binding domain (NBD) 1 with characteristic Walker motifs A and B. However, experimental studies concerning this proposed topology (2-5) remain controversial. According to its sequence, P-glycoprotein is classified as a member of a large family of membrane transporters known as the ATP-binding cassette superfamily that includes yeast, bacteria, and mammalian transporters (6, 7). P-glycoprotein is proposed to function as an ATP-driven efflux pump, transporting through the plasma membrane an unusually broad but well defined spectrum of structurally unrelated cytotoxic drugs, including the Vinca alkaloids, anthracyclines, epipodophyllotoxins, and taxanes (8 -10).A large body of evidence suggests that the transmembrane domains of the P-glycoprotein participate in the recognition of substrates, whereas the ATP hydrolysis necessary for transport is carried out by both NBD regions with a similar efficiency in an alternating fashion (11-16). The mechanism of coupling ATP hydrolysis at the two cytoplasmic nucleotidebinding sites to drug transport by the intramembrane drugbinding site(s) is likely to involve substantial conforma...

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Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.