Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1

Membrane transporters of the adenine nucleotide binding cassette (ABC) supe rfamily utilize two either identical or homologous nucleotide binding domai ns (NBDs). Although the hydrolysis of ATP by these domains is believed to d rive transport of solute, it is unknown why two rather than a single NBD is required. In the well studied P-glycoprotein multidrug transporter, the tw o appear to be functionally equivalent, and a strongly supported model prop oses that ATP hydrolysis occurs alternately at each NBD (Senior, A. E., al- Shawi, M. H., and Urbatsch, I. L. (1995) FEBS Lett 377, 285-289), To assess how applicable this model may be to other ABC transporters, we have examin ed adenine nucleotide interactions with the multidrug resistance protein, M RP1, 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 A DP trapping predominantly at NBD2, Despite this apparent asymmetry in the t wo 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 allosteric ally enhance the binding of ATP at NBD1. Glutathione, which supports drug t ransport by the protein, does not enhance ATP binding but stimulates the tr apping of ADP, Thus MRP1 may employ a more complex mechanism of coupling AT P utilization to the export of agents from cells than P-glycoprotein.