Functional Interactions Between Nucleotide Binding Domains and Leukotriene C₄Binding Sites of Multidrug Resistance Protein 1 (ABCC1)

Abstract

Multidrug resistance protein 1 (MRP1) is a member of the "C" branch of the ATP-binding cassette transporter superfamily. The NH 2 -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. We have 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 cysteinyl leukotriene C 4 (LTC 4 ) . Overall, the results support a model in which occupancy of both NBD1 and NBD2 by ATP results in the formation of a low-affinity conformation of the protein. However, nonhydrolyzable ATP analogs (␤,␥-imidoadenosine 5Ј-triphosphate and adenylylmethylene diphosphonate) failed to substitute for ATP or adenosine 5Ј-O-(thiotriphosphate) (ATP␥S) in decreasing LTC 4 photolabeling. Furthermore, mutations of the signature sequence in either NBD that had no apparent effect on azido-ATP binding abrogated the 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 LTC 4 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.

Converting Nonhydrolyzable Nucleotides to Strong Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Agonists by Gain of Function (GOF) Mutations

“…Their precise roles are unclear but probably relate to their participation in controlling NBD dimerization and/or signaling between the ATP binding pockets and the translocation pathway (39,40). The G551D mutation in the NBD1 signature sequence abolishes ATP-dependent CFTR gating and causes severe CF.…”

Portrait of multifaceted transporter, the multidrug resistance-associated protein 1 (MRP1/ABCC1)

“…On the other hand, substitution of the conserved Gly in the ABC signature sequence of NBD1 for Ala completely inactivates the protein, while this mutation in NDB2 causes only a partial inactivation [121].…”

“…Inactivation of any one of the two ATP-binding sites results in the complete loss of trapping activity of the protein [101]. In contrast, chemical modifications or mutations of the consensus motifs in the two NBDs of MRP1 have different effects on the transport [43,53,121,178]. Mutations of key residues in the Walker A or Walker B of NBD1 decreased LTC 4 transport activity to 30-50% of the wild-type protein, whereas the corresponding mutations in NBD2 essentially inactivated the protein [43,53,121].…”

“…Conversely, the trapping of ADP at NBD2 has also been shown to enhance the ATP binding at NBD1 in one report [53], while other studies indicated that mutations affecting nucleotide binding and trapping at NDB2 had no effect on ATP binding at NBD1 [43,121].…”

“…High-and low-affinity drug binding states of MRP1 were also distinguished. Either ATP with or without vanadate, or the poorly hydrolyzable ATP analog, ATPγS, can result in the transition from a high-to a low-affinity state [121]. Prolonged binding of either ATP or ADP in NBD2 effectively locked the protein in a low-affinity substrate binding state, while increased ADP trapping in NBD1 prevented this transition [122].…”