Multidrug resistance protein MRP1 reconstituted into lipid vesicles: secondary structure and nucleotide-induced tertiary structure changes.
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.
Manciu
L
L
Laboratoire de Chimie-Physique des Macromolécules aux Interfaces (LCPMI), Université Libre de Bruxelles, B-1050 Brussels, Belgium.
Chang
X B
XB
Riordan
J R
JR
Ruysschaert
J M
JM
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D003903:Deuterium / Q000378:metabolism; D018432:Drug Resistance, Multiple; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D003903:Deuterium; D008081:Liposomes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate)