Dynamics of P-Glycoprotein: A Fluorescence and Disulfide Cross-Linking Approach

Abstract

P-glycoprotein (Pgp), a member of the ABC transporter family, couples ATP hydrolysis to efflux of hydrophobic molecules including drugs used in chemotherapy. Here we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two NBDs are undergoing. Cysteines introduced into equivalent regions in the N- and C-terminal NBDs of cysteine-less mouse Pgp were labeled with fluorescent dyes for ensemble and single molecule FRET (smFRET) spectroscopy with lipid-reconstituted Pgp. In ensemble fluorescence experiments, adding substrate and/or nucleotide increased the FRET efficiency for all mutants to varying degrees, suggesting that the two NBDs approach one another upon substrate binding. Analysis of smFRET data suggests that the NBDs of Pgp alternate between at least two major conformations, a low and a high FRET state, during verapamil stimulated ATP hydrolysis. Vanadate inhibition shifted the population toward the high FRET state while the Pgp inhibitor cyclosporin resulted in a shift towards the low FRET state. Furthermore, we have utilized direct disulfide cross-linking to test whether complete dissociation of the NBDs is required for ATPase stimulation by bulky drug molecules. Tethering the two NBDs together at their C-terminal ends did not abolish drug stimulated ATP activity, indicating the motion the NBDs are undergoing is mainly on the level of the catalytic sites, with the C-terminal ends of the NBDs acting as a hinge. Taken together, the data provide an indication as to what the nature and magnitude of the structural rearrangements are that Pgp is undergoing during the catalytic cycle.