Molecular Model and ATPase Activity of Carboxyl-Terminal Nucleotide Binding Domain from Human P-Glycoprotein

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Feng Qian, Dongzhi Wei^*, Jianglan Liu, and Shengli Yang

State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, P. R. China; fax: 8621-64250068; E-mail: dzhwei@ecust.edu.cn

^* To whom correspondence should be addressed.

Received October 8, 2004; Revision received December 28, 2004

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ATP binding and hydrolysis are required for P-glycoprotein mediated multidrug resistance. To investigate the molecular mechanism involved in ATP binding and hydrolysis, a three-dimensional model of the carboxyl-terminal nucleotide binding domain (NBD2) was built by homology modeling. Modeling revealed the human P-glycoprotein ATP-binding site and the possible role of conserved Gln1118 residue. Recombinant NBD2 was overexpressed in Escherichia coli and the conserved Gln1118 residue was mutated to an alanine residue. The V_max for ATP hydrolysis by the mutant NBD2 was ~56% of the V_max of wild-type NBD2. But both proteins displayed similar affinity for ATP, with K_m of 479 and 466 µM for mutant and wild-type NBD2, respectively. These results suggest that the possible role of Gln1118 is as an activating residue for ATP hydrolysis. The molecular model also provided structural information about the interactions between NBD2 and the chemosensitizer quercetin. The complex indicated that quercetin was tightly bound to the ATP-binding site and competed for binding. The three-dimensional model of NBD2 can be used to both guide enzymological studies and provide a theoretical basis for the design of potential multidrug resistance reversers.

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KEY WORDS: molecular modeling, multidrug resistance, nucleotide binding domain, P-glycoprotein, site-directed mutagenesis

DOI: 10.1134/S0006297906130037

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