title: Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1.
abstract: Nucleotide Binding Domains (NBDs) are responsible for the ATPase activity of the multidrug resistance protein 1 (MRP1). A series of NBD1-linker-NBD2 chimeric fusion proteins were constructed, expressed and purified, and their ATPase activities were analyzed. We report here that a GST linked NBD1(642-890)-GST-NBD2(1286-1531) was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (K(m)=2.17 mM, V(max)=12.36 nmol/mg/min), which was comparable to the purified and reconstituted MRP1. In contrast, neither a mixture of NBD1 and GST-NBD2 nor the NBD1-GST-NBD1 fusion protein showed detectable ATPase activity. Additionally, the E1455Q mutant was found to be nonfunctional. Measurements by both MIANS labeling and circular dichroism spectroscopy revealed significant conformational differences in the NBD1-GST-NBD2 chimeric fusion protein compared to the mixture of NBD1 and GST-NBD2. The results suggest a direct interaction mediated by GST between the two NBDs of MRP1 leading to conformational changes which would enhance its ATPase activity.
authors.affiliation: National Laboratory of Biomacromolecules, Center for structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, PR China.
authors.forename: Lixin
authors.initials: L
authors.lastname: Wan
authors.forename: Xingjie
authors.initials: X
authors.lastname: Liang
authors.forename: Youguo
authors.initials: Y
authors.lastname: Huang
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D000251:Adenosine Triphosphatases; D017931:DNA Primers; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011993:Recombinant Fusion Proteins; C421109:multidrug resistance-associated protein 1