Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1.
Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs). Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289). To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein. Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2. Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping. Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1. Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP. Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein.
Hou
Y
Y
Mayo Foundation, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259, USA.
Cui
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L
Riordan
J R
JR
Chang
X
X
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002478:Cells, Cultured; D006224:Cricetinae; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019920:Photoaffinity Labels; D011485:Protein Binding; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000227:Adenine Nucleotides; D001386:Azides; D019920:Photoaffinity Labels; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D004317:Doxorubicin; D000255:Adenosine Triphosphate