ABC Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-throughput Screening Journal Article


Authors: Iram, S. H.; Gruber, S. J.; Raguimova, O. N.; Thomas, D. D.; Robia, S. L.
Article Title: ABC Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-throughput Screening
Abstract: Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a "2-color MRP1" construct by fusing GFP and TagRFP to MRP1 nucleotide binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. 2-color MRP1 transport activity was normal, as shown by vesicular transport of [3H]E217betaG and doxorubicin efflux in AAV-293 cells. We quantified fluorescence resonance energy transfer (FRET) from GFP to TagRFP as an index of NBD conformational changes. Our results show that ATP binding induces a large-amplitude conformational change that brings the NBDs into closer proximity. FRET was further increased by substrate in the presence of ATP, but not by substrate alone. The data suggest that substrate binding is required to achieve a fully closed and compact structure. ATP analogs bind MRP1 with reduced apparent affinity, inducing a partially closed conformation. The results demonstrate the utility of the 2-color MRP1 construct for investigating ABC transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1.
Keywords: Cardiovascular Research Institute; ABC efflux transporters; Fluorescence techniques; Membrane targets; Optical spectroscopy (fluorescence, DC, etc.); Pharmacokinetics, metabolism and activation; Resistance; Structure-activity relationships and modeling; Structure/function/mechanism
Journal Title: Molecular pharmacology
Volume: 88
Issue: 1
ISSN: 1521-0111; 0026-895X
Publisher: Unknown  
Date Published: 2015
Start Page: 84
End Page: 94
Language: ENG
DOI/URL:
Notes: LR: 20150501; CI: The American Society for Pharmacology and Experimental Therapeutics.; JID: 0035623; OTO: NOTNLM; aheadofprint