Functionally Similar Vanadate-induced 8-Azidoadenosine 5′-[α -³²P]Diphosphate-trapped Transition State Intermediates of Human P-glycoprotein are Generated in the Absence and Presence of ATP Hydrolysis

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump whose overexpression confers multidrug resistance to cancer cells. Pgp exhibits a robust drug substrate-stimulable ATPase activity, and vanadate (Vi) blocks this activity effectively by trapping Pgp nucleotide in a non-covalent stable transition state conformation. In this study we compare Vi-induced [α-³²P]8-azido-ADP trapping into Pgp in the presence of [α-³²P]8-azido-ATP (with ATP hydrolysis) or [α- ³²P]8-azido-ADP (without ATP hydrolysis). Vi mimics P_i to trap the nucleotide tenaciously in the Pgp·[α- ³²P]8-azido-ADP·Vi conformation in either condition. Thus, by using [α-³²P]8-azido-ADP we show that the Vi-induced transition state of Pgp can be generated even in the absence of ATP hydrolysis. Furthermore, half-maximal trapping of nucleotide into Pgp in the presence of Vi occurs at similar concentrations of [α-³²P]8-azido-ATP or [α-³²P]8-azido-ADP. The trapped [α- ³²P]8-azido-ADP is almost equally distributed between the N- and the C-terminal ATP sites of Pgp in both conditions. Additionally, point mutations in the Walker B domain of either the N- (D555N) or C (D1200N)-terminal ATP sites that arrest ATP hydrolysis and Vi-induced trapping also show abrogation of [α-³²P]8-azido-ADP trapping into Pgp in the absence of hydrolysis. These data suggest that both ATP sites are dependent on each other for function and that each site exhibits similar affinity for 8-azido-ATP (ATP) or 8-azido-ADP (ADP). Similarly, Pgp in the transition state conformation generated with either ADP or ATP exhibits drastically reduced affinity for the binding of analogues of drug substrate ([¹²⁵I]iodoarylazidoprazosin) as well as nucleotide (2′(3′)-O-(2,4,6-trinitrophenyl)-adenosine 5′-triphosphate). Analyses of Arrhenius plots show that trapping of Pgp with [α-³²P]8-azido-ADP (in the absence of hydrolysis) displays an ∼2.5-fold higher energy of activation (152 kJ/mol) compared with that observed when the transition state intermediate is generated through hydrolysis of [α-³²P]8-azido-ATP (62kJ/mol). In aggregate, these results demonstrate that the Pgp·[α- ³²P]8-azido-ADP (or ADP)·Vi transition state complexes generated either in the absence of or accompanying [α- ³²P]8-azido-ATP hydrolysis are functionally indistinguishable.