ATP-Binding Cassette 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 "two-color MRP1" construct by fusing green fluorescent protein (GFP) and TagRFP to MRP1 nucleotide-binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. Two-color MRP1 transport activity was normal, as shown by vesicular transport of [(3)H]17β-estradiol-17-β-(D-glucuronide) 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 two-color MRP1 construct for investigating ATP-binding cassette transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1.

Fig. 1.

Expression, localization, and FRET of two-color MRP1. (A) Schematic diagram of two-color MRP1 construct showing an intrasequence GFP (two amino acid linker on each side) and a C-terminus TagRFP (five amino acid linker). (B) Crystal structure of mouse P-gp in apo condition showing the separated NBDs (left) (Aller et al., 2009); crystal structure of nucleotide-bound Staphylococcus aureus Sav1866 showing the closed NBD structure (right) (Dawson and Locher, 2006). (C) Immunoblots of whole-cell lysates (10 μg) containing wild-type (WT-MRP1) and two-color MRP1 (2C-MRP1) compared with untransfected cells (control), with detection by mouse monoclonal anti-MRP1 (1:5000 dilution, 1 hour at room temperature) or anti-GFP antibody (1:2000 dilution, 1 hour at room temperature). (D) AAV-293 cells expressing two-color MRP1 were plated on glass-bottom chambered coverslips as described in experimental procedures. Fluorescent images were collected using a Nikon inverted microscope equipped with a 60× objective. Widefield lamp excitation and computer-controlled filter wheels were used to acquire GFP and TagRFP images. (E) MRP1 membrane expression and lateral diffusion investigated by TIRF microscopy and FRAP of a 1.3-μm target region. (F) Data from FRAP were analyzed using a nonlinear curve fit with a single exponential function. (G) MRP-1 FRET did not depend on protein expression level.

Fig. 2.

Functional characterization of two-color MRP1. (A) ATP-dependent uptake of [³H]E₂17βG by the membrane vesicles. Membrane vesicles were incubated with substrate and transport reaction was allowed for 1 minute at 37°C. Values are mean ± S.D. for triplicate determinations in a single experiment; both wild-type (WT-MRP1) and two-color MRP1 (2C-MRP1) values were significantly different from control (P < 0.05). Similar results were obtained in a second experiment with vesicles derived from independent transfection. (B) AAV-293 cells (control) and two-color MRP1-expressing cells were exposed to 10 μM DOX for 15 minutes. DOX-containing media was replaced with PBS, and cells were imaged using a confocal microscope equipped with 63× objective. DOX and GFP were excited at 488-nm wavelength using an argon laser, with emission bands of 565–650 nm for DOX and 496–533 nm for GFP. DOX was detected by red fluorescence and was mostly localized in the nucleus; two-color MRP1 (green) showed plasma membrane localization. To inhibit MRP1 activity, cells were incubated with MK571 (50 μM) or BMH (1 mM) for 15 minutes before DOX treatment. All error bars represent mean ± S.E.

Fig. 3.

Dynamic FRET changes of two-color MRP1 in permeabilized AAV-293 cells. FRET measurements were made as described in the experimental procedures. Cells plated on glass-bottom–chambered coverslips were permeabilized for 2 minutes at room temperature in 50 μg/ml saponin in PBS, which washed out the endogenous cell contents including the nucleotides. PBS buffer supplemented with 5 mM MgCl₂ was used in all FRET experiments. All incubations (10–15 minutes) and FRET measurements were made at room temperature. (A) FRET increased with ATP. (B) MRP1 FRET response to nucleotide and transportable substrates E₂17βG or cysteinyl leukotriene (LTC₄). (C) MRP1 interaction with ATP analogs applied at 1 mM concentration. (D) Competition of 1 mM nucleotide analogs with 500 μM ATP. Analogs did not prevent ATP from increasing MRP1 FRET. (E) ATP analogs increased MRP1 FRET when applied at high concentration. (F) The effect of inhibitors of MRP1, including 1 mM vanadate, 50 μM MK571, and 1 mM BMH. With the exception of the apo condition, 1 mM ATP + 50 μM E₂17βG were added to each. ATP, ATP+ E₂17βG, ATP+LTC₄, and ADP values were statistically significant versus apo (P < 0.05). All error bars represent mean ± S.E. AMP-PCP, adenylylmethylenediphosphonate.

Fig. 4.

High-throughput screening with two-color MRP1. (A) Hits from the National Clinical Collection. (B) We observed significantly less variability and improved signal to noise when screening with two-color MRP1 on the basis of fluorescence lifetime compared with fluorescence intensity. (C) Dose response for select hit compounds. Epigallocatechin gallate showed the highest apparent affinity (EC₅₀ = 1.7 μM). (D) DOX uptake into the nucleus of human embryonic kidney cells after pretreatment with candidate compounds. Values are mean nuclear fluorescence ± S.E., normalized to untransfected cells in the same microscopic field. The red horizontal line indicates a high level of DOX accumulation (as in untransfected cells), the green line indicates decreased accumulation of DOX suggesting drug efflux by MRP1. *P < 0.0001 versus untransfected; *P < 0.0001 versus “+MRP1”. All error bars represent mean ± S.E.