The relationship between MRP1 activities and its NBD conformational changes.
MIANS, a sulfhydryl-reactive fluorescence, was used to label the cysteines of MRP1 (multidrug resistance protein), and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS, which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment. The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs, of which one lies close to NBD1, and the other near NBD2. ATP, ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS. The collisional quenchers, acrylamide, l-, and Cs+ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged. Both MIANS and NEM, which are sulfhydryl-reactive reagents, inhibited MRP1 ATPase activity, whereas anticancer drugs activated it. These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1. Nucleotides can bind directly to NBDs, but drugs may react first with TMDs, which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity, which is coupled with the transport of its substrates. Taken together, the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1.
Huang
Zhenhua
Z
National Laboratory of Biomacromolecules, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Huang
Youguo
Y
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002586:Cesium / Q000737:chemistry; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D008956:Models, Chemical; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D000817:Anilino Naphthalenesulfonates; D002586:Cesium; D003545:Cysteine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C421109:multidrug resistance-associated protein 1