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Characterization of the ATPase activity of a novel chimeric fusion protein consisting of the two nucleotide binding domains of MRP1 |
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| Authors: | Lixin Wan Xingjie Liang |
| Institution: | a National Laboratory of Biomacromolecules, Center for structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, PR China b Graduate School of the Chinese Academy of Sciences, Bejing 100049, PR China c Laboratory of Nanobiomedicine and Nanosafety, Division of Nanomedicine and Nanobiology, National Center for Nanosciences and Technology of China, Beijing 100190, PR China |
| 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 NBD1642-890-GST-NBD21286-1531 was able to hydrolyze ATP at a rate of about 4.6 nmol/mg/min (Km = 2.17 mM, Vmax = 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. |
| Keywords: | MRP1 Multidrug resistance Nucleotide binding domains Domain-domain interaction ATPase activity Conformation |
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