Role of glycine-534 and glycine-1179 of human multidrug resistance protein (MDR1) in drug-mediated control of ATP hydrolysis.
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.
Szakács
G
G
National Institute of Haematology and Immunology, Membrane Research Group of the Hungarian Academy of Sciences, Daróczi ut 24, H-1113 Budapest, Hungary.
Ozvegy
C
C
Bakos
E
E
Sarkadi
B
B
Váradi
A
A
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D001693:Biological Transport, Active; D018432:Drug Resistance, Multiple / Q000235:genetics*; D014644:Genetic Variation; D005998:Glycine / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D005998:Glycine