Received for publication October 22, 2004.
Revised January 11, 2005.
Accepted for publication March 8, 2005.

Functional Interactions Between Nucleotide Binding Domains and LTC₄ Binding Sites of Multidrug Resistance Protein 1 (ABCC1)

Abstract

MRP1 is a member of the 'C' branch of the ATP-binding cassette (ABC) transporter superfamily. The NH₂-proximal nucleotide binding domain (NBD1) of MRP1 differs functionally from its COOH-proximal domain (NBD2). NBD1 displays intrinsic high-affinity ATP binding and little ATPase activity. In contrast, ATP binding to NBD2 is strongly dependent on nucleotide binding by NBD1 and NBD2 is more hydrolytically active. Previously, we demonstrated that occupancy of NBD2 by ATP or ADP, markedly decreased substrate binding by MRP1. We have further explored the relationship between nucleotide and substrate binding by examining the effects of various ATP analogs and ADP trapping, as well as mutations in conserved functional elements in the NBDs, on the ability of MRP1 to bind the photoactivatable, high-affinity substrate, leukotriene C₄ (LTC₄). Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in formation of a low-affinity conformation of the protein. However, non-hydrolyzable ATP analogs (AMP-PNP and AMP-PCP) failed to substitute for ATP or ATP--S in decreasing LTC4 photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding, abrogated formation of a low-affinity substrate binding state in the presence of ATP or ATP--S. We suggest that the effect of these mutations, and possibly the failure of some ATP analogs to decrease LTC₄ binding, may be attributable to an inability to elicit a conformational change in the NBDs that involves interactions between the signature sequence and the -phosphate of the bound nucleotide.

Key words: Organic anion, Leukotrienes, Resistance

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