Conformational Changes of the Multidrug Transporter P-Glycoprotein in Solution and Lipid Discs

Abstract

P-glycoprotein (Pgp) exports hundreds of chemically unrelated, hydrophobic compounds out of cells. Since Pgp can greatly affect bioavailability, pharmacokinetics and efficacy of therapeutic drugs, there is great interest in understanding the mechanism by which drugs are extruded. Pgp is a prototype ABC transporter with two nucleotide binding domains (NBDs) that bind and hydrolyze ATP, leading to extensive conformational changes across the transmembrane domains that result in substrate translocation across the cell membrane. According to current alternating access models, binding of ATP promotes NBDs dimerization (outward-facing conformation), and ATP-hydrolysis leads to their dissociation (inward-facing conformation). Despite decades of biochemical/biophysical studies, the mechanism by which the NBDs control these conformational changes is still controversial. We have used luminescence resonance energy transfer (LRET) to measure distances between the two NBDs during the ATP hydrolysis cycle. Pgp was labeled with the LRET probes, reconstituted in a lipid bilayer nanodisc, and the distance between the NBDs was measured at 37°C in nucleotide-free, ATP-bound, MgATP hydrolysis conditions. Pgp in nanodiscs showed increased basal ATPase activity and robust verapamil-stimulated ATP hydrolysis. Our LRET results suggest that the NBDs associate and dissociate in response to ATP binding and hydrolysis. Like the bacterial homolog MsbA, Pgp showed two main conformations (dimeric NBDs and dissociated NBDs) that coexist in equilibrium. However, the proportion of molecules in each conformation and the ATP-hydrolsysis driven changes are distinctly different suggesting that Pgp predominantly resides in the dissociated NBD state. The results are consistent with a recent snapshots analysis at ambient temperature by negative stain electron microscopy. The impact on substrate translocation mechanisms in Pgp vs. MsbA will be discussed. The LRET studies open new possibilities of studying conformational changes at physiological temperatures under native-like conditions.