A hexahistidine-tagged C-terminal nucleotide-binding domain (H 6 -NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein.

ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Delé age, G., Di Pietro, A., and Jault, J. M. 32 P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu 504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.