Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione.
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.
Manciu
Liliana
L
Structure and Function of Biological Membranes-Center of Structural Biology and Bioinformatics, Free University of Brussels, B-1050 Brussels, Belgium.
Chang
Xiu-Bao
XB
Buyse
Frédéric
F
Hou
Yue-Xian
YX
Gustot
Adelin
A
Riordan
John R
JR
Ruysschaert
Jean Marie
JM
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000577:Amides / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D006224:Cricetinae; D018432:Drug Resistance, Multiple; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D007668:Kidney; D008024:Ligands; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014162:Transfection; D014357:Trypsin
D000255:Adenosine Triphosphate; D000577:Amides; D005978:Glutathione; D008024:Ligands; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010446:Peptide Fragments; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D014357:Trypsin; C421109:multidrug resistance-associated protein 1