The human multidrug resistance protein (MDR1) (P-glycoprotein), a member of the ATP-binding cassette (ABC) family, causes multidrug resistance by an active transport mechanism, which keeps the intracellular level of hydrophobic compounds below a cell-killing threshold. Human MDR1 variants with mutations affecting a conserved glycine residue within the ABC signature of either or both ABC units (G534D, G534V, G1179D and G534D/G1179D) were expressed and characterized in Spodoptera frugiperda (Sf9) cell membranes. These mutations caused a loss of measurable ATPase activity but still allowed ATP binding and the formation of a transition-state intermediate (nucleotide trapping). In contrast with the wild-type protein, in which substrate drugs accelerate nucleotide trapping, in the ABC signature mutants nucleotide trapping was inhibited by MDR1-substrate drugs, suggesting a miscommunication between the drug-binding site(s) and the catalytic domains. Equivalent mutations of the two catalytic sites resulted in a similar effect, indicating the functional equivalence of the two sites. On the basis of these results and recent structural information on an ABC–ABC dimer [Hopfner, Karcher, Shin, Craig, Arthur, Carney and Tainer (2000) Cell 101, 789–800], we propose a key role of these glycine residues in the interdomain communication regulating drug-induced ATP hydrolysis.
Abbreviations used: ABC, ATP-binding cassette; BeFx, beryllium fluoride (the exact composition of beryllium fluoride is not known); HisP, histidine permease (ATPase subunit of the maltose-transport system of enterobacteria); MDR1, human multidrug resistance protein; Rad50cd, catalytic domain (a dimer of the ATPase subunits) of Rad50; RbsA, ATPase subunit of the ribose-transport system of enterobacteria; Sf9 cells, Spodoptera frugiperda ovarian cells.