Model of coupled antiport and uncoupled proton leak through EmrE.

(A) All of the drug- and proton-bound states that are reasonably populated at near physiological temperature and pH and the transitions between these states observed by NMR leads to a model for EmrE transport that allows for both coupled antiport (orange) and proton leak (red solid line). (B) In WT-EmrE, the C-terminal tail on the open face acts as a secondary gate (top), minimizing proton leak in the absence of substrate. Truncation of EmrE in Δ107-EmrE removes this gate (bottom). (C) The Drug binding to a secondary binding site near the tail opens the gate (top), allowing proton exit from the primary binding site near E14, and drug to progress to the primary binding site at E14. This leads to either coupled antiport (A, orange) as shown. If the substrate does not rapidly move into the primary binding site, only proton entry/exit occurs upon opening of the secondary gate, resulting in drug-gated proton leak (A, red dashed line). Truncation of the C-terminal tail in Δ107-EmrE (bottom) allows uncoupled proton leak in the absence of substrate (A, red solid line).

C-terminal tail truncation does not impair the ability of EmrE to confer resistance to toxic compounds.

(A-C) WT-, E14Q-, or Δ107-EmrE was heterologously expressed in MG1655-Δemre E. coli using a plasmid with p15 origin and pTrc promoter without induction to minimize any growth defect due to expression. In vivo growth assays were monitored by OD700 to allow consistent monitoring in the absence (B) or presence of (C) ethidium bromide. Growth at 15 hours (A) shows identical growth for WT-EmrE and Δ107-EmrE in the presence of ethidium, while E14Q-EmrE is severely impaired (A,B). There is a 20% reduction in growth for Δ107-EmrE relative to WT-EmrE or non-functional EmrE (p < 0.001), but this does not prevent the mutant from transporting ethidium out of the cell and thus conferring resistance (A,C). The error bars show the standard deviation across six replicates (two biological replicates with three technical replicates each). All p-values were calculated from a two-sided t-test. (*) p < 0.05 (**) p < 0.01 (***) p < 0.001.

C-terminal tail truncation enhances proton leak.

(A-B) Pyranine fluorescence directly reports on proton leak through EmrE. (A) WT (black), Δ107 (red, to distinguish in vitro assays from the cellular assays of Fig. 2) or E14Q-EmrE (gray) proteoliposomes with 1 mM internal pyranine and internal pH 6.5 were diluted 100-fold into pH 7.5 buffer (solid lines) or pH 7.5 buffer with CCCP (dashed lines) and fluorescence was normalized to time zero. CCCP is a protonophore, providing a positive control for maximal proton leak under these conditions. (B) Pyranine fluorescence normalized by subtracting the fluorescence of proteolipsomes diluted into pH 6.5 (no gradient, baseline) from the fluorescence of proteoliposomes diluted into pH 7.5 (transport) shows intraliposomal pH change with proteoliposomes in the lag time prior to initial fluorescence read and increased intraliposomal pH change for Δ107-EmrE than WT-EmrE or E14Q-EmrE. (C-E) Solid supported membrane electrophysiology data shows measurable charge movement through WT- and Δ107-EmrE proteoliposomes in the presence of a pH gradient alone, as compared to empty liposomes, with increased charge transport through Δ107-EmrE. (C) Current is recorded in real time as a matching pH internal buffer (pH 6.5) is flowed over the liposomes to establish baseline, then a higher pH (pH 7) buffer is flowed over the liposomes to create an outwardly directed proton gradient (dashed box), and finally the initial buffer (pH 6.5) is flowed back over the liposomes to reverse the charge movement and return to baseline. (D) The recorded current during the period of the applied gradient (dashed box, C) is integrated to determine the transported charge during that time. In all cases, Δ107-EmrE shows increased proton leak compared to WT-EmrE and controls. The error bars show the standard deviation across three replicates or sensors.

The pH dependence of alternating access in Δ107-EmrE is distinct from WT-EmrE.

TROSY-HSQC spectra of Δ107-EmrE in the absence (A) and presence (B) of the tight-binding ligand tetraphenylphosphonium (TPP). While drug binding slows the dynamics of the protein at both low (red) and high (blue) pH, as evident by the better spectral quality in B, in both drug-free and drug-bound Δ107-EmrE the dynamics of the mutant are highly sensitive to the pH conditions. ZZ-Exchange Spectroscopy of Δ107-EmrE bound to of TPP was used to quantify the alternating-access rates at low and high pH. ZZ-exchange spectra with the indicated delays are shown for (C) pH 5.5 and (D) pH 7.7. (E) The composite peak intensity ratios for F78, G80, R82, L83 and R106 fit to an exchange rate of 4±1 s-1 at pH 5.5. At pH 7.7, the composite peak intensity ratios for G80, R82, L83 and R106 fit to an exchange rate of 17±3 s-1.

The C-terminus tail caps the water wire from the open side.

(A) Logarithm of the minimum water distance log(S) histogram. (B-D) The following panels illustrate a few snapshots in the simulation. The membrane normal vector points to the open side of EmrE. Two dashed arrows show the ligand pathway and the water chain respectively. TM1 to TM3 in subunit B is shown transparently to better illustrate the interface between the two subunits. (B) Dry snapshot of WT-EmrE. (C) Wet snapshot of Δ107-EmrE. (D) A rare event snapshot when WT-EmrE is hydrated. The color codes are the same as in Figure 6. Yellow stars highlight the backbone of the C-terminal residue (R106 or H110) and the yellow arrowhead (B, D) highlights the backbone of R106 in the full length construct to illustrate where the tail would terminate in Δ107.

The structural basis of the C-terminus gating.

(A) The minimum distance between side chain hydrogens for A61A, I68B, and I71B. In WT-EmrE, the sidechain of A61A is significantly closer to I68B and I71B, while the distance between I68B and I71B does not change significantly. The error bars show the standard deviation along the trajectory. All p-values were calculated from a two-sided t-test, (**) p < 0.01 (***) p < 0.001 (B) The proton transport potential of mean force (PMF), as a function of the distance between the center of the excess charge (CEC) and the donor (E14) on the direction of transport (See Eq.1 in Methods). (C) A snapshot of the transition state. The orange sphere is the proton CEC. (D) Conformations of the A61A, I68B, I71B triad from two different angles. The membrane normal shown at the right points to the open side of EmrE. The upper panels are from a side view, and the lower panels are looking top-down into the primary binding site from the open side. The transparent surface in the upper panels shows the water wire. Unlabeled residues shown as stick representation are E14B, Y60A, and S64A.

pH titration of TPP+-bound Δ107-EmrE supports the possibility of secondary gating.

(A) In WT-EmrE bound to TPP+, one E14 residue and one H110 residue, are the only titratable sites (dark red circles labeled H+). In Δ107-EmrE, H110 is not present, suggesting that only one titratable group should remain (E14). (B) The proton and nitrogen chemical shifts for individual residues of TPP+-bound Δ107-EmrE were recorded as a function of pH. The resulting titration profiles do not show the expected single-pKa pattern. Some are curved, consistent with multiple pKa values, and others are consistent with a single pKa but at either high or low pH. All of the data can be globally fit to two pKa values, using either a 2-pKa fit (5.6 and 7.1, grey) or single pKa fit at the relevant value (5.6, red; 7.1, blue). (C) Residues sensitive to each pKa value are plotted on the faRM model (21) using the indicated color scale. (D) Residues strongly sensing the lower pKa value cluster around the C-terminus (R106) and 3-4 loop (includes residue D84) on both the open and closed face of the transporter, while the 1-2 loop (includes residue E25) and T56 on the open side of the pore sense both pKa values (Left).

Intrinsic leak in Δ107-EmrE does not synergize with harmane-induced leak.

SSME traces of transported charge corresponding to proton leak in the absence (A) and presence (B) of harmane show that 16 µm harmane induces leak in WT-EmrE that is comparable to the leak observed through Δ107-EmrE in the absence of harmane. In the presence of increasing concentrations of harmane (C) the leak signal for WT-EmrE quickly converges to that of Δ107-EmrE. The leak observed for Δ107-EmrE is more variable, displaying larger standard deviations than WT-EmrE proteoliposomes (C). This could be due to greater variability in the unregulated transport activity of Δ107-EmrE compared to harmane-gated leak in WT-EmrE, and the impact of this unregulated behavior on the sensitivity of SSME to variation in the absolute number of proteoliposomes adsorbed on the surface sensor. Growth assays in the absence of substrate (Fig. 1A) show a clear growth defect for E. coli expressing Δ107-EmrE compared to WT-EmrE, which is nearly eliminated when cells are grown in the presence of 25 µM harmane (E-F). Δ107-EmrE data is shown in blue for cellular assays and red for in vitro assays to readily distinguish the assay type. The error bars show the standard deviation across 3 sensors for SSME or across six replicates for growth assays (two biological replicates with three technical replicates each). All p-values were calculated from a two-sided t-test (*) p < 0.05 (**) p < 0.01 (***) p < 0.001.