Drug, Nucleotide, and Lipid Interactions with P-glycoprotein

Project Summary
P-glycoprotein pumps drugs, xenobiotics and nutrients out of cells via a partially characterized
ATP-dependent mechanism. Due to the extreme substrate promiscuity of P-gp, it contributes to
the disposition of nearly all small molecule drugs and to drug-drug interactions. P-gp may be
particularly important in cancer cell drug resistance due to its over expression in several
cancers. The aims of this proposal are to fill knowledge gaps in three distinct aspects of P-gp
mechanism. Each aim shares the common mechanistic element of conformational dynamics.
The first aim is to define the P-gp conformations at low ATP occupancy in order to understand
how they control downstream conformational changes that are essential for substrate efflux, and
how they are different in the presence vs. the absence of substrates. These conformational
differences are hypothesized to be essential for minimizing wasteful expenditure of ATP in the
absence of transportable substrate and to control proper conformational access in downstream
steps. These differences will be identified by rapid, pre-equilibrium, kinetic methods with state-
of-the-art kinetic modeling and Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX) with
P-gp in lipid nanodiscs.
The second aim is to understand the extreme sensitivity of P-gp to its lipid environment. The
lipid nanodisc platform provides fine control of the lipid bilayer in which P-gp resides. P-gp will
be incorporated in nanodiscs with varying ratios of lipids with different head groups or acyl
chains that provide different surface charge of bilayer fluidity. The basal- and substrate
dependent ATPase activity in the different lipid environments will be correlated with changes in
conformation determined by H/DX.
The third aim is to determine the mechanism by which P-gp is able to interact with such a wide
range of substrates and inhibitors. Substrate promiscuity is an increasingly appreciated behavior
for many proteins throughout biology but the molecular basis is poorly defined. Here, pre
equilibrium binding kinetics and single molecule fluorescence methods will be used to compare
the distribution of binding parameters in P- gp ensembles vs. single P-gp nanodiscs in order to
determine whether P-gp exploits interconverting conformations or persistent distinct
conformations to recruit and retain drugs of varying structure.

Grant Number: 5R01GM144446-04
NIH Institute/Center: NIH
Principal Investigator: WILLIAM ATKINS