Abstract
A key element of the structural model of ABC-ATP-ases 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 gamma-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 nucleotideas 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.
Highlights
Overexpression of the human multidrug resistance-associated protein (ABCC1) MRP1, a member of the ATP-binding cassette (ABC)1 family, has been suggested to be responsible for cancer drug resistance [1]
A general feature of ABC proteins is that they utilize the energy of MgATP binding and hydrolysis for conformational motion
In ABC transporters, in which the catalytic ABC domains are attached to membrane-spanning transmembrane domains, the conformational transitions result in the transport of substrates across biological membranes [31]
Summary
Overexpression of the human multidrug resistance-associated protein (ABCC1) MRP1, a member of the ATP-binding cassette (ABC) family, has been suggested to be responsible for cancer drug resistance [1]. The structure of Rad50cd, a bacterial ABC-ABC ATPase, represents two functionally interacting ABC subunits, dimerizing in a head-to-tail orientation [11] In this dimer, the two ABC domains complement each other’s active sites, forming composite catalytic centers. Comparison of the structures of the ATP-free and ATP-bound Rad50cds revealed a nucleotide-mediated movement of the lobe including the LSGGQ motif toward the bound ATP molecule within the opposite subunit [11] This movement, which might be a general feature in ABC proteins, may provide the structural requirement for the completion of the catalytic site. On the basis of the experiments, the authors concluded that the signature sequence of one ABC unit is adjacent to the Walker A motif of the opposing ABC [16] They studied the effect of drug binding on crosslinking in the same experimental system [17]. These results demonstrate that drug binding to the transmembrane domains can induce conformational changes within the ABC domains
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