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J. Biol. Chem., Vol. 278, Issue 46, 46052-46063, November 14, 2003

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Functional Importance of Polar and Charged Amino Acid Residues in Transmembrane Helix 14 of Multidrug Resistance Protein 1 (MRP1/ABCC1)

IDENTIFICATION OF AN ASPARTATE RESIDUE CRITICAL FOR CONVERSION FROM A HIGH TO LOW AFFINITY SUBSTRATE BINDING STATE^*

Da-Wei Zhang, Hong-Mei Gu, Donna Situ¶, Anass Haimeur||, Susan P. C. Cole¶**, and Roger G. Deeley¶

From the Division of Cancer Biology and Genetics, Cancer Research Institute, and the Departments of ^¶Pathology & Molecular Medicine, Biochemistry, and Anatomy & Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu¹⁰⁸⁹ in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn¹¹⁰⁰, Ser¹⁰⁹⁷, and Lys¹⁰⁹², which are all predicted to be on the same face of the helix as to Glu¹⁰⁸⁹, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys¹⁰⁹², increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17-estradiol 17-(-D-glucuronide) (E₂17G) but not cysteinyl leukotriene 4 (LTC₄), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp¹⁰⁸⁴ that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E₂17G and LTC₄, despite the fact that LTC₄ binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp¹⁰⁸⁴ may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.

Received for publication, July 31, 2003 , and in revised form, September 3, 2003.

^* This work was supported in part by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Run and by Grant MOP-10519 from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Recipient of a Canadian Institutes of Health Research postdoctoral award.

^** Canada Research Chair in Cancer Biology and Senior Scientist of Cancer Care Ontario.

Stauffer Research Professor of Queen's University and Director, Division of Cancer Biology and Genetics, Cancer Research Inst., Queen's University. To whom correspondence should be addressed: Cancer Research Inst., Suite 300, 10 Stuart St., Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2979; Fax: 613-533-6830; E-mail: deeleyr{at}post.queensu.ca.

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