Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC 4 . The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.Multidrug resistance-associated protein 1 (MRP1), 1 a member of the ATP-binding cassette transporter family, confers resistance to a wide range of chemotherapeutic drugs including anthracyclines, vinca alkaloids, and epipodophyllotoxins by exporting them out of cells (1-6). ATP binding and hydrolysis as well as the presence of GSH is essential for transport to occur (2,3,(7)(8)(9)(10)(11)(12)(13)(14). In addition, MRP1 transports anionic compounds such as LTC 4 (8, 15), bilirubin glucuronide (16), glucuronide, and sulfate-conjugated estrogens (17, 18) and bile salts (19), glutathione disulfide (20), and arsenical and antimonial oxyanions (2).Its predicted topology is characteristic of a typical ABC transporter: a core structure containing two membrane-spanning domains (MSD1 and MSD2), each of them composed of six transmembrane (TM) segments and two nucleotide-binding domains (NBD1 and NBD2) that are located at the cytoplasmic face of the membrane (6, 21-23). In addition to this co...
Multidrug resistance protein MRP1 is an ATP-dependent drug efflux pump that confers resistance in human cancer cells to various chemotherapeutic drugs. We have reconstituted purified MRP1 in lipid vesicles. The reconstituted protein conserves ATPase and drug transport activity. Structural analysis of MRP1 was investigated by infrared spectroscopy for the first time. This technique offers a unique opportunity to determine structural parameters characterizing a membrane protein in its lipid environment. Addition of different ligands (MgATP, MgATPgammaS, MgADP and P(i), and MgADP) did not significantly affect the MRP1 secondary structure, which is made of 46% alpha-helix, 26% beta-sheet, 12% beta-turns, and 17% random coil. Binding of MgATP increased the protein accessibility to the solvent, suggesting a modification in the tertiary organization of the protein. Hydrolysis of MgATP to MgADP and P(i) did not significantly change the global accessibility of the protein. Release of P(i), after hydrolysis, caused a decrease in the accessibility of MRP1 to the water phase which brings the protein back to its initial conformation. All together, the data demonstrate that MRP1 adopts different structures during its catalytic cycle.
Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug-resistant cells. On the contrary, three compounds (4P P-deoxy-4P P-iodo-doxorubicin, 4-demethoxydaunorubicin and 3P P-(3-methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/ MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1-mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.-M. (2000) Biochemistry 39, 13026^13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug-dependent conformational changes. Binding of drugs (4-demethoxy-daunorubicin and 3P P-(3-methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process. ß
Analytical chemistry Z 0400 Structure, Orientation, and Conformational Changes in Transmembrane Domains of Multidrug Transporters -[37 refs.]. -(VIGANO, C.; MANCIU, L.; RUYSSCHAERT*, J.-M.; Acc. Chem. Res. 38 (2005) 2, 117-126; Lab. Struct. Func. Biol. Membr., Univ. Libre Bruxelles, B-1050 Brussels, Belg.; Eng.) -Lindner 20-298
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