Conformational Changes During the ATP Hydrolysis Cycle of the Multidrug Transporter P-Glycoproteinin Response to Substrate Binding

Abstract

P-glycoprotein (Pgp) is a multidrug transporter consisting of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) that mediates the efflux of a wide variety of compounds, including many chemotherapeutic agents. Here, we assessed the conformational changes elicited by substrates and inhibitors on Pgp reconstituted in nanodiscs and studied at 37°C using luminescence resonance energy transfer (LRET). We used a fully-functional Pgp mutant (N607C/T1252C) that contained only two cysteines, introduced in the NBDs for labeling with the LRET probes (donor: Tb3+ chelate-maleimide; acceptor: Bodipy FL-maleimide). We were able to detect differences in the conformational changes on the NBDs side in response to binding of substrates and inhibitors to the TMDs. In the state closer to physiologic, during continuous hydrolysis in the presence of the substrates verapamil, valinomycin or taxol, the maximal NBDs separation between the closed (NBD dimers) and open conformations (more separated NBDs) was 13-15 Å, with ∼50% of molecules in the closed conformation, similar to the vanadate-inhibited state. In the presence of the inhibitors tariquidar or zosuquidar the distance between the NBDs in the open conformation was longer. These results suggest that the open-conformation distance is shorter in the presence of substrates, when ATPase activity is higher, whereas inhibition is associated with a larger separation of the NBDs under ATP hydrolysis conditions. This work was supported in part by the Cancer Prevention and Research Institute of Texas Grant RP101073, National Institute of Health Grants RGM102928 and R01GM118594, and the South Plains Foundation from Lubbock, TX.