Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis.

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg‚ADP caused a reversible decrease in the binding capacity of the transported substrate ( 3 H)-vinblastine and the nontransported modulator ( 3 H)XR9576 to P-gp in CH r B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-A-S also caused a reduction in the binding capacity of ( 3 H)-vinblastine but not for the modulator ( 3 H)XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.