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, 88 (1), 475-82

Investigation of Ligand Binding to the Multidrug Resistance Protein EmrE by Isothermal Titration Calorimetry

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Investigation of Ligand Binding to the Multidrug Resistance Protein EmrE by Isothermal Titration Calorimetry

Curtis W Sikora et al. Biophys J.

Abstract

Escherichia coli multidrug resistance protein E (EmrE) is an integral membrane protein spanning the inner membrane of Escherichia coli that is responsible for this organism's resistance to a variety of lipophilic cations such as quaternary ammonium compounds (QACs) and interchelating dyes. EmrE is a 12-kDa protein of four transmembrane helices considered to be functional as a multimer. It is an efflux transporter that can bind and transport cytoplasmic QACs into the periplasm using the energy of the proton gradient across the inner membrane. Isothermal titration calorimetry provides information about the stoichiometry and thermodynamic properties of protein-ligand interactions, and can be used to monitor the binding of QACs to EmrE in different membrane mimetic environments. In this study the ligand binding to EmrE solubilized in dodecyl maltoside, sodium dodecyl sulfate and reconstituted into small unilamellar vesicles is examined by isothermal titration calorimetry. The binding stoichiometry of EmrE to drug was found to be 1:1, demonstrating that oligomerization of EmrE is not necessary for binding to drug. The binding of EmrE to drug was observed with the dissociation constant (K(D)) in the micromolar range for each of the drugs in any of the membrane mimetic environments. Thermodynamic properties demonstrated this interaction to be enthalpy-driven with similar enthalpies of 8-12 kcal/mol for each of the drugs in any of the membrane mimetics.

Figures

FIGURE 1
FIGURE 1
EmrE uses energy from the proton gradient across the inner membrane of Escherichia coli to efflux quaternary ammonium compounds from the cytoplasm to periplasm as shown. Extramembrane (EM) loops and N- and C-termini are labeled for clarity. The topology was established by Son et al. (2003).
FIGURE 2
FIGURE 2
Representative titration calorimetry of EmrE in SUVs with ethidium. (A) Each peak corresponds to the injection of 8 μl of 0.5 mM ethidium in SUVs into the reaction cell containing 40 μM EmrE in SUVs. The concentration of E. coli polar lipid that formed SUVs in this experiment was 37.5 mg/ml. (B) Cumulative heat of reaction is displayed as a function of the injection number. The solid line is the least-squares fit to the experimental data of separate trials (indicated by symbols ×, ▪, and □). It corresponds with a KD of 5.5 μM. (C) Linearization of the data in a single trial in a Scatchard plot as an alternative way of measuring the KD.
FIGURE 3
FIGURE 3
Representative titration calorimetry of EmrE in SUVs with methyl viologen. (A) Each peak corresponds to the injection of 8 μl of 0.500 mM methyl viologen in SUVs into the reaction cell containing 40 μM EmrE in SUVs. The concentration of E. coli polar lipid that formed SUVs in this experiment was 37.5 mg/ml. (B) Cumulative heat of reaction is displayed as a function of the injection number. The solid line is the least-squares fit to the experimental data of separate trials (indicated by symbols ×, ▪, and □). It corresponds with a KD of 38.2 μM. (C) Linearization of the data in a single trial in a Scatchard plot as an alternative way of measuring the KD.
FIGURE 4
FIGURE 4
(A) Thermogram of EtBr binding to SUVs with a KD of 0.58 mmol. Each peak corresponds to the injection of 8 μl of 40 mM EtBr solution into the reaction cell containing SUVs. The concentration of E. coli polar lipid that formed SUVs in this experiment was 17.5 mg/ml. (B) Cumulative heat of reaction is displayed as a function of the injection number. The solid line is the least-squares fit to the experimental data points. It corresponds with a KD of 0.58 mM. (C) Linearization of the data in a single trial in a Scatchard plot as an alternative way of measuring the KD.
FIGURE 5
FIGURE 5
Structures of lipophilic cations used in this study.

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