Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy

Brandy Verhalen,Stefan Ernst,Michael Börsch,Stephan Wilkens

doi:10.1074/jbc.m111.301192

Abstract

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10-100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.

Highlights

P-glycoprotein is an ATP-binding cassette transporter involved in multidrug resistance
To directly monitor the structural changes Pgp is undergoing during ATP hydrolysis-driven drug translocation, we designed a fluorescence resonance energy transfer (FRET) spectroscopy-based system to monitor structural changes in real time via fluorescent dyes attached to cysteine residues introduced into equivalent regions of the N- and C-terminal nucleotide binding domains (NBDs) using site-directed mutagenesis
Site-directed Mutagenesis—To address domain movement in Pgp using FRET spectroscopy, pairs of cysteines were placed at equivalent positions in the N- and C-terminal nucleotide binding domains of the transporter for covalent labeling with maleimide-linked fluorescent dyes

Summary

Background

P-glycoprotein is an ATP-binding cassette transporter involved in multidrug resistance. According to the current mechanistic model for ABC transporter function [11, 21], Pgp can exist in at least two major conformations during the catalytic cycle as follows: an “inward”-facing conformation, with the drug binding pocket exposed to the cytoplasmic side and separated NBDs, and an “outward”-facing conformation with a low affinity drug-binding site exposed to the extracellular space and closely interacting NBDs. Conversion of the inward- to outward-facing conformation requires binding of MgATP, whereas subsequent ATP hydrolysis and/or product release involving one (or both) NBD resets the transporter to the ground state (the inward-facing conformation). To directly monitor the structural changes Pgp is undergoing during ATP hydrolysis-driven drug translocation, we designed a FRET spectroscopy-based system to monitor structural changes in real time via fluorescent dyes attached to cysteine residues introduced into equivalent regions of the N- and C-terminal NBDs using site-directed mutagenesis.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION