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. 2016 Mar;12(3):141-5.
doi: 10.1038/nchembio.1999. Epub 2016 Jan 11.

Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE

Anindita Gayen  1 , Maureen Leninger  1 , Nathaniel J Traaseth  1
Affiliations

Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE

Anindita Gayen et al. Nat Chem Biol. 2016 Mar.

Abstract

Secondary active transport proteins play a central role in conferring bacterial multidrug resistance. In this work, we investigated the proton-coupled transport mechanism for the Escherichia coli drug efflux pump EmrE using NMR spectroscopy. Our results show that the global conformational motions necessary for transport are modulated in an allosteric fashion by the protonation state of a membrane-embedded glutamate residue. These observations directly correlate with the resistance phenotype for wild-type EmrE and the E14D mutant as a function of pH. Furthermore, our results support a model in which the pH gradient across the inner membrane of E. coli may be used on a mechanistic level to shift the equilibrium of the transporter in favor of an inward-open resting conformation poised for drug binding.

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Figures

Figure 1
Figure 1. pH-induced conformational changes to EmrE
Overlay of 1H/13C SOFAST-HMQC NMR spectra for Ile methyl groups at the indicated pH values for (a) wild-type and (c) E14D EmrE. Arrows indicate the direction of chemical shift changes from low to high pH. (b, d) Normalized chemical shift perturbations at 25 °C for wild-type and E14D EmrE, respectively. The global fit to individual sites gave the indicated apparent pKa values of 7.0 and 5.0. The error bars reflect the standard deviation among residues used to construct the fit. (e) E. coli resistance assays using serial 10-fold dilutions in the presence of an LB agar plate with 240 µM ethidium bromide and carbenicillin. The assays were performed at pH 7.0 and 4.7 using wild-type EmrE, E14D, and a control that contained no expressed EmrE. Control dilution experiments in the absence of ethidium are shown in Supplementary Figure 13. The resistance assay was repeated with similar results at least five times.
Figure 2
Figure 2. Glu-14 protonation sets the resting state
(a) Schematic of the alternating access model. The vectorial transport rules forbid conformational exchange in the fully unloaded transporter. (b) Conformational exchange dynamics probed with PUREX in aligned bicelles at the two indicated pH values. (c) Quantified exchange rates as a function of pH (error bars reflect a 90% confidence interval). All of the PUREX curves used to construct panel c are shown in Supplementary Figure 5. (d) Overlay of a methyl T1zz experiment (red) and HSQC spectrum (black) of deprotonated EmrE (pH 9.15). The dotted boxes connect the two exchanging peaks and suggest the presence of conformational dynamics in the Glu-14 deprotonated transporter. (e) Tryptophan fluorescence of EmrE in DMPC vesicles under different pH combinations. (f) Disruption of the ΔpH vesicles with addition of DDM detergent. The same fluorescence result was obtained in three independent trials.
Figure 3
Figure 3. Allosteric conformational change from Glu-14 protonation
(a) Heat map of the chemical shift perturbations between pH=5.5 and pH=9.15 mapped onto the Cα atoms of EmrE (3B5D). Green spheres denote residues that became too weak to be followed in the pH titration below ~7.0. (b) Perturbation data for E14D EmrE mapped onto the same structure (difference between pH=3.75 and pH=7.5). Both perturbations datasets are viewed from the perspective of the open side of the transporter. (c) Cartoon model illustrating the proposed role of the latch in stabilizing the open conformation of EmrE, which is consistent with the NMR perturbations in the pH titration, analysis of the crystal structure of EmrE, and the loss of resistance for I54G. Note that the TM domains are indicated with numbers. (d) E. coli resistance assays carried out using serial 10-fold dilutions on an LB agar plate (pH=7) containing carbenicillin in the absence (top) and presence (bottom) of 240 µM ethidium bromide. The plasmid contained wild-type EmrE, I54G, I54L, or a control vector where EmrE was not expressed. The resistance assay was repeated twice with the same result.
Figure 4
Figure 4. Conformational bias from loop-2 mutant in mixed EmrE dimers
1H/13C SOFAST-HMQC experiments of (a) wild-type and (b) I54L EmrE in the presence of TPP+. (c and d) Mixed dimer of wild-type and I54L where the 13C enriched protein is underlined. The mixed sample where I54L was NMR enriched gave more intense peaks for monomer “b” whereas the labeled wild-type gave greater peak intensities for monomer “a”. Insets within the HMQC spectra show 1D slices for Ile-101 to illustrate the skewed populations. The asterisks indicate lipid peaks.
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