The Role of the Conserved Glycines of ATP-binding Cassette Signature Motifs of MRP1 in the Communication between the Substrate-binding Site and the Catalytic Centers

Abstract

A key element of the structural model of ABC-ATPases is the interaction of the two ABC domains. They complement each other's active sites in a way that the ABC signature motif (LSGGQ) of one subunit interacts with the ␥-phosphate of the ATP, bound at the Walker motifs of the opposite subunit. In the present study, the conserved glycines in the fourth position of the LSGGQ motifs of human MRP1 were substituted for aspartic acids (G771D and G1433D), the mutants were expressed in Sf9 insect cells, and the nucleotide-as well as the transported substrate-protein interactions were studied. We found that these transport-and ATPase-incompetent mutants showed no nucleotide trapping under any of the conditions examined. However, when measuring the effect of nucleotide and transported substrates on the vanadate-induced cleavage reactions, we found that the effect of substrates on the cleavage reactions was significantly different in the mutant MRP1 proteins than in the wild type. Although the transported substrates (e.g. etoposide ؉ oxidized glutathione) stimulated the formation of the posthydrolytic complex in the wild type, this reaction was inhibited in the signature mutants. Our study also revealed that a similar mutation in the ABC signature of either ABC unit resulted in the same effect. We suggest that the conserved glycine residues in both LSGGQ segments are part of the conformational network, which is responsible for the accelerated hydrolytic activity upon interaction of the protein with its transported substrates. This intramolecular communication between the substrate-binding site and the catalytic centers is assumed to be a general feature of the molecular mechanism of ABC transporters.

ATP-driven MalK Dimer Closure and Reopening and Conformational Changes of the “EAA” Motifs Are Crucial for Function of the Maltose ATP-binding Cassette Transporter (MalFGK2)

“…4B). This result provides further evidence for the ABC signature being involved in communication between NBDs and TMDs (24,(43)(44)(45)(46).…”

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts

“…It is worth reminding here that inhibition of the PI3K/Akt pathway enhances in vitro sensitivity of AML blasts to etoposide, 45 which is a well-established MRP1 substrate. 46 This has been interpreted mainly as a consequence of downregulated NF-kB activity, but it could not be ruled out that it was owing to decreased drug efflux by MRP1.…”

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

“…A recent study from our laboratory suggests that the ABC signature motif is a candidate for intramolecular communication [157]. The structures of ATP-bound NBD dimers show that the conserved second Gly residue in the ABC signature motif interacts with the oxygen of the gamma-phosphate of the ATP, bound to the Walker motif of the opposite subunit [118].…”