Journal of Biological Chemistry
Volume 292, Issue 50, 15 December 2017, Pages 20412-20424
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Molecular Biophysics
Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer–associated P-glycoprotein during ATP hydrolysis

https://doi.org/10.1074/jbc.M117.814186Get rights and content
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P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD–NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

ABC transporter
fluorescence resonance energy transfer (FRET)
membrane
membrane bilayer
multidrug transporter
spectroscopy
LRET
luminescence resonance energy transfer
nanodisc

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This work was supported in part by the Cancer Prevention and Research Institute of Texas Grant RP101073, National Institute of Health Grants RGM102928 and R01GM118594, and the South Plains Foundation from Lubbock, TX. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This article contains supplemental Figs. S1–S8.

1

Both authors contributed equally to this work.

2

Present address: School of Natural Sciences, University of California, Merced, 4225 N. Hospital Rd., Atwater, CA 95301.

3

Present address: Amgen Inc., Mail Stop ASF1-2101A, 1120 Veterans Blvd., South San Francisco, CA 94080.

4

Present address: Dept. of Natural Sciences, Lubbock Christian University, Lubbock, TX 79407.