Biophysical and Structural Approaches to Characterize the Mechanism of P-glycoprotein, a Multidrug Efflux Transporter

Abstract

P-glycoprotein (P-gp), an ATP Binding Cassette transporter (ABCB1), plays an important role in multidrug resistance in cancers and clinical drug interactions. P-gp utilizes the energy from ATP hydrolysis to drive conformational transitions from the inward-facing (IF) to outward-facing (OF) states that lead to extracellular drug release. In addition to the nucleotide-dependent conformational rearrangements which provide alternating access of substrates to intracellular or extracellular milieu, the drug binding sites have been proposed to cycle between high and low affinities to facilitate drug binding or dissociation. However, drug-specific effects on nucleotide-dependent affinity changes to P-gp remain largely uncharacterized. The lack of binding kinetics in general also limits the mechanistic interpretation of P-gp transport. Furthermore, molecular details of IF and OF conformations have not been demonstrated because the structure of OF P-gp remains unavailable by classical methods. To address these questions, this dissertation describes the work in employing complementary biophysical, biochemical and structural techniques to gain additional insight on P-gp. By using Fluorescence Correlation Spectroscopy to monitor fluorescent probe affinities for P-gp, our results indicate that changes in affinities are both probe- and nucleotide-dependent. Combined with a novel antibody-based quenching strategy for vesicle-based transport, we find that Flutax-2 may represent an under-appreciated class of P-gp substrates whereby transport is uncoupled to nucleotide binding and hydrolysis. Hydrogen-Deuterium Exchange Mass Spectrometry was used to characterize the detailed structural dynamics of P-gp in the IF or OF states, and in detergent or nanodiscs environment. Vanadate inhibition of P-gp to promote the OF conformations leads to an overall decrease in deuterium uptake throughout the NBDs, but also causes an increased solvent exposure of a peptide from TM1 which supports the OF conformation with an opening of drug binding sites towards extracellular space. Interestingly, several peptides displaying EX1 exchange kinetics indicate that P-gp motions occur on a wide range of time scale, from seconds to hours. Lastly, Surface Plasmon Resonance is presented as viable technique for studying drug interactions with membrane proteins reconstituted into nanodiscs. Experimental considerations, advantages and disadvantages associated with different biosensor chips are discussed, and successful preliminary experiments with small-molecules binding to P-gp nanodiscs are demonstrated. Together, these results shed light on the complex structural and functional mechanism of P-gp.