Role of Carboxylate Residues Adjacent to the Conserved Core Walker B Motifs in the Catalytic Cycle of Multidrug Resistance Protein 1 (ABCC1)

Abstract

MRP1 belongs to subfamily "C" of the ABC transporter superfamily. The nucleotide-binding domains (NBDs) of the C family members are relatively divergent compared with many ABC proteins. They also differ in their ability to bind and hydrolyze ATP. In MRP1, NBD1 binds ATP with high affinity, whereas NBD2 is hydrolytically more active. Furthermore, ATP binding and/or hydrolysis by NBD2 of MRP1, but not NBD1, is required for MRP1 to shift from a high to low affinity substrate binding state. Little is known of the structural basis for these functional differences. One minor structural difference between NBDs is the presence of Asp COOH-terminal to the conserved core Walker B motif in NBD1, rather than the more commonly found Glu present in NBD2. We show that the presence of Asp or Glu following the Walker B motif profoundly affects the ability of the NBDs to bind, hydrolyze, and release nucleotide. An Asp to Glu mutation in NBD1 enhances its hydrolytic capacity and affinity for ADP but markedly decreases transport activity. In contrast, mutations that eliminate the negative charge of the Asp side chain have little effect. The decrease in transport caused by the Asp to Glu mutation in NBD1 is associated with an inability of MRP1 to shift from high to low affinity substrate binding states. In contrast, mutation of Glu to Asp markedly increases the affinity of NBD2 for ATP while decreasing its ability to hydrolyze ATP and to release ADP. This mutation eliminates transport activity but potentiates the conversion from a high to low affinity binding state in the presence of nucleotide. These observations are discussed in the context of catalytic models proposed for MRP1 and other ABC drug transport proteins.ATP-binding cassette (ABC) 1 transporters are ubiquitous transmembrane proteins that couple ATP hydrolysis to the energy-dependent transport of a wide variety of endogenous and exogenous molecules across biological membranes. Multidrug resistance protein (MRP) 1 (ABCC1) belongs to the "C" subfamily of the ABC superfamily and was discovered by virtue of its ability to cause multidrug resistance when overexpressed in a human small cell lung cancer cell line (1, 2). The MRP1 multidrug resistance phenotype is similar to that resulting from overexpression of P-glycoprotein (P-GP), and involves resistance to many relatively hydrophobic, natural product type, cytotoxic agents. However, unlike P-GP, MRP1 can also transport various structurally unrelated organic anionic conjugates, including: glutathione, glucuronide, and sulfate conjugates, such as the potent mediator of inflammation, cysteinyl leukotriene LTC 4 , the cholestatic glucuronide-conjugated estrogen E 2 17␤G, the sulfate conjugate estrone 3-sulfate, and the glutathione epoxide conjugate of the highly mutagenic aflatoxin B1 (3-10).Typically, the functional form of ABC proteins consists of two hydrophilic nucleotide-binding domains (NBDs) located at the cytoplasmic surface of the membrane and two hydrophobic transmembrane spanning domains (MSD) that are thought to for...

Mutational Analysis of Ionizable Residues Proximal to the Cytoplasmic Interface of Membrane Spanning Domain 3 of the Multidrug Resistance Protein, MRP1 (ABCC1)

“…transporter (36,37). Thus, inactivation of NBD2 abolishes transport by MRP1, but inactivation of NBD1 results in only a partial loss of activity.…”

“…Our demonstration that vanadate-induced trapping of azido-ADP by the mutant E1204L protein (and specifically by NBD2) was substantially increased suggests that the mutation may impair the ability of NBD2 to release ADP after hydrolysis of ATP, which could in turn impair substrate translocation and/or release. Alternatively, the E1204L mutant may hydrolyze ATP and release ADP very rapidly in the absence of vanadate but be unable to proceed through a second catalytic cycle (36). In effect, the mutation may diminish the coupling of the catalytic activity of MRP1 to transport in a way that affects some substrates more than others.…”

Identification and Characterization of Functionally Important Elements in the Multidrug Resistance Protein 1 COOH-terminal Region

“…32 P]ADP labeling was performed as described previously (39). Sf21 membrane vesicles (20 g of protein) were incubated with 5 M 8-azido-[␣-…”

“…P]ATP at 37°C-ADP orthovanadate and beryllium fluoride trapping was performed as described previously (33,39). Briefly, Sf21 membrane vesicles (20 g of protein) were incubated with of 8-azido-[␣-…”

“…The gels were dried for 2 h at 80°C before audioradiography. 4 in the presence of ATP, ATP and Orthovanadate, or ATP␥S-Photoaffinity labeling of MRP fragments by LTC 4 in the presence of nucleotides has been described previously (24,39). Briefly, membrane vesicles (50 g) were incubated with either ATP (4 mM), ATP (4 mM), and orthovanadate (1 mM) or with ATP␥S (4 mM) with 5 mM MgCl 2 in TB for 30 min at room temperature, followed by incubation with 200 nM [ 3 H]LTC 4 for 30 min at room temperature.…”