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

• Published in: The Journal of biological chemistry 2012-01, Vol.287 (2), p.1112-1127
• Main Authors: Verhalen, Brandy, Ernst, Stefan, Börsch, Michael, Wilkens, Stephan
• Format: Article
• Language: English
• Subjects: ABC Transporter; Adenosine Triphosphate - chemistry; Adenosine Triphosphate - genetics; Adenosine Triphosphate - metabolism; Animals; ATP Binding Cassette Transporter, Subfamily B - chemistry; ATP Binding Cassette Transporter, Subfamily B - genetics; ATP Binding Cassette Transporter, Subfamily B - metabolism; ATPases; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer (FRET); Ligands; Membrane Biology; Membrane Proteins; Membrane Transport; Mice; Models, Molecular; Multidrug Transporters; Protein Conformation; Single Molecule Biophysics; Vanadates - chemistry; Vanadates - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.301192

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. Background: P-glycoprotein is an ATP-binding cassette transporter involved in multidrug resistance. Results: The two nucleotide binding domains are found to be in close association during the catalytic cycle as determined by fluorescence spectroscopy. Conclusion: Small distance changes were observed during ATP hydrolysis supporting an alternating site mechanism. Significance: Understanding the mechanism of P-glycoprotein is pertinent for developing inhibitors aimed at overcoming multidrug resistance.