Inducing conformational preference of the membrane protein transporter EmrE through conservative mutations

Prior to investigating whether mutations in one subunit of an EmrE dimer could induce a change in the conformational equilibrium in vitro, NMR experiments were carried out on homodimer samples of wild-type EmrE and mutants (L51I, I62L). EmrE samples were prepared in magnetically aligned bicelles consisting of a 3.5/1 molar ratio of long chain (O-14:0-PC) and short chain (6:0-PC) phospholipids at a pH value of 5.8 that corresponds to the Glu14 proton-bound form of EmrE. Solid-state NMR experiments were carried out using the PISEMA experiment (Wu et al., 1994), since this technique gives the largest frequency separation between peaks corresponding to the two monomers within the asymmetric dimer (Gayen et al., 2013). PISEMA spectra of homodimer samples of wild-type, L51I, and I62L selectively labeled with ¹⁵N-tyrosine are shown in Figure 1B. Each spectrum displayed 10 peaks, which confirms the presence of twice the number of peaks as tyrosine residues in the primary sequence of EmrE (Tyr4, Tyr6, Tyr40, Tyr53, Tyr60), and is consistent with previous observations (Gayen et al., 2013; Morrison et al., 2012; Gayen et al., 2016; Dutta et al., 2014b).

To analyze whether heterodimers might lead to a preferred conformation, we prepared mixtures of wild-type EmrE with the L51I or I62L mutant. In each experiment, only the wild-type or mutant was isotopically enriched with ¹⁵N-tyrosine and mixed with its partner protein at natural abundance. Using this approach, only one protein was NMR active, while the other was NMR silent. PISEMA spectra corresponding to wild-type/L51I or wild-type/I62L where wild-type was isotopically enriched showed a primary set of intense peaks that corresponded to monomer B (Figure 2A). These signals were superimposable onto those peaks in the wild-type EmrE PISEMA spectrum (Figure 2—figure supplement 1A). Next, we carried out the reverse experiments where the mutants were isotopically enriched and wild-type was NMR silent. In these spectra, the isotopically enriched mutant (L51I or I62L) in the mixed dimer also showed an intense set of peaks (Figure 2B), yet these signals did not overlap with those of isotopically enriched wild-type in the mixed dimer (Figure 2—figure supplement 1B). The peak positions did however match onto the corresponding peaks in the L51I and I62L homodimer PISEMA spectra (Figure 2—figure supplement 1A). These data indicate that wild-type EmrE has a preference for monomer B in the heterodimer while L51I or I62L prefers monomer A for the proton-bound form of EmrE (Figure 2C). It is important to note that the mixture of wild-type EmrE and the L51I or I62L mutant produces a statistical fraction of heterodimers and homodimers in the samples. While our analysis focused on the most intense set of signals, we were able to resolve a weaker set of signals that likely corresponded to homodimers at a lower contour level (Figure 2—figure supplement 2). Nevertheless, these observations demonstrate that a minimal heterodimer comprised of a single conservative mutation can strongly influence the conformational equilibrium and perturb the energy landscape of EmrE.

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All experiments in Figure 2 were performed on the proton-bound and drug-free form of EmrE. However, substrate transport requires EmrE to sample additional conformations within its catalytic cycle. To investigate how other states might impact the conformational equilibrium, we turned to solution NMR spectroscopy since it is a more sensitive technique to probe structure and conformational dynamics. To confirm the observations from PISEMA experiments in Figure 2, we collected solution NMR experiments of mixed dimers under proton-bound sample conditions in ²H-14:0-PC/²H-6:0-PC (1/2 mol/mol) isotropic lipid bicelles. The mixed samples of wild-type EmrE and L51I were prepared in a similar manner as for solid-state NMR experiments with two notable differences: (1) the NMR active protein was isotopically enriched with ¹³C at the C^δ position of isoleucine residues and (2) the ratio of isotopically labeled wild-type EmrE to L51I at natural abundance was 1/3. The latter was possible due to the increased sensitivity of methyl detection in solution NMR spectroscopy. Figure 3A shows the ¹H/¹³C HMQC spectrum of isotopically enriched wild-type EmrE in a mixed dimer sample with L51I. From this spectrum, we observed an intense set of signals corresponding to monomer B, which confirms the results of our solid-state NMR experiments in aligned lipid bicelles. Thus, under sample conditions used to collect solution and solid-state NMR spectra (i.e. isotropic and aligned bicelles, respectively), the skewed conformational equilibrium is preserved.

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Next, we sought to investigate whether other states of the transport cycle, such as deprotonation of Glu14 and drug bound forms, may alter the conformational equilibrium. Previously, we reported pH induced chemical shift perturbations within EmrE’s NMR methyl spectrum that were centered at a pH value of 7.0 and corresponded to a biologically relevant pK_a of Glu14 (Gayen et al., 2016). Thus, we acquired a ¹H/¹³C HMQC spectrum with a mixed sample of ¹³C enriched EmrE and natural abundance L51I at a pH value of 9.1. Similar to the proton-bound dataset, this spectrum showed intense peaks that corresponded to the signals stemming from monomer B (Figure 3B). This is in stark contrast to the spectrum of wild-type EmrE at high pH that showed approximately equal intensities of monomer A and B peaks (Figure 3B). This result supports the conclusion that the skewed conformational equilibrium is preserved under conditions in which Glu14 is deprotonated. Note that we also observed monomer A signals at a lower contour level, which arises due to the statistical nature of mixing as discussed above (Figure 3—figure supplement 1A). Using the relative peak intensities of A and B signals in the spectrum, we estimated the equilibrium population of wild-type EmrE to assume monomer B in the heterodimer at ~96% (range of 90% to 99% based on standard deviation). This value corresponds to a free energy of ~1.8 kcal/mol induced by the L51I mutation in the heterodimer.

To determine the effect of drug binding on the conformational equilibrium, we acquired NMR spectra for the mixed dimers in the presence of ethidium and tetraphenylphosphonium. These drug substrates are commonly used in the EmrE literature for measuring resistance, transport, and binding (Yerushalmi et al., 1995; Curnow et al., 2004; Robinson et al., 2018). Addition of ethidium induced a significant amount of spectral broadening and ablation of peak intensities in both the wild-type sample and that of the mixed dimer where wild-type was isotopically enriched (Figure 3C). These data suggest intermediate chemical exchange, which likely stems from motion corresponding to exchange between inward-open and outward-open states (Cho et al., 2014). From these data, it is likely that the equilibrium is less skewed in the mixed sample upon ethidium binding. Namely, if the populations were maintained to the same extent as in the mixed dimer sample at pH 9.1 (Figure 3B), the effect of intermediate chemical exchange would not induce peak broadening beyond detection. Hence, the effect of ethidium binding suggests that the conformational bias is reduced when the heterodimer is bound to a drug substrate. Furthermore, the line-broadening supports the presence of drug-induced dynamics in the heterodimer, which would enable conformational exchange needed for drug transport.

Finally, we investigated the effect of tetraphenylphosphonium binding on the conformational equilibrium. Unlike ethidium, tetraphenylphosphonium binds with greater affinity to EmrE and significantly reduces the rate of conformation exchange (i.e. slow chemical exchange regime) (Cho et al., 2014; Morrison and Henzler-Wildman, 2014). We carried out the same NMR experiment by adding tetraphenylphosphonium to a mixed dimer sample of ¹³C^δ-Ile labeled EmrE mixed with natural abundance L51I (Figure 3D). Unlike the effect of ethidium binding, the spectra were nicely resolved in the presence of tetraphenylphosphonium. We observed that monomer B peaks were more intense relative to those of monomer A; however, the signal intensities stemming from monomer A were significantly greater compared to any of the drug-free samples (see I58^A, I62^A, and I88^A peaks in Figure 3D). Specifically, the ratio of monomer B to A peak intensities were reduced upon addition of tetraphenylphosphonium relative to the deprotonated sample. Using the relative peak intensities of A and B, we estimated the equilibrium population of wild-type to assume monomer B in the heterodimer at ~86% (standard deviation range of 79% to 92%), which corresponds to ~1.1 kcal/mol induced by the single mutant in the heterodimer. This result is in agreement with the ethidium binding experiment. These data also provide evidence that drug binding would not trap the transporter into a single conformation in the transport cycle and is consistent with previous work proposing the substrate controls the rate of conformational exchange (Morrison and Henzler-Wildman, 2014).

E. coli growth inhibition assays against ethidium bromide were carried out to determine the biological significance of our NMR observations. Initially, we tested whether the mutations L51I and I62L were able to confer resistance when expressed individually. Figure 4A shows a growth inhibition assay using serial 10-fold dilutions on an LB agar plate with wild-type, L51I, I62L, and a control vector. From these data, L51I and I62L displayed a strong phenotype toward ethidium and were indistinguishable relative to wild-type EmrE. Thus, each mutation appears to be fully functional and able to couple to the electrochemical potential to accomplish active drug efflux.

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To probe the effect of heterodimer induced changes to the conformational equilibrium, we designed transporter complementation experiments by expressing two genes: (1) gene 1 consisted of wild-type EmrE, L51I, or I62L and (2) gene 2 consisted of single topology variants of EmrE. The latter utilized similar constructs as previous work showing insertion topology can be controlled by mutating arginine and lysine residues within the primary sequence (Rapp et al., 2007) (i.e. the positive inside rule; Heijne, 1986). Using this technology, gene 2 consisted of N- and C-termini facing the cytoplasmic direction (EmrEⁱⁿ) or periplasmic direction (EmrE^out), respectively. Resistance assays verified literature results (Rapp et al., 2007) that EmrEⁱⁿ and EmrE^out expressed individually were unable to confer a phenotype to ethidium, whereas co-expression displayed a strong phenotype toward this compound (Figure 4B).

Next, we carried out resistance assays using mixtures of wild-type, L51I, or I62L with EmrEⁱⁿ or EmrE^out. The experiments where wild-type EmrE was co-expressed with EmrEⁱⁿ or EmrE^out revealed identical levels of conferred resistance relative to wild-type EmrE alone (Figure 5A). To ensure that wild-type was forming a heterodimer with EmrEⁱⁿ or EmrE^out, we designed a control experiment in which an additional mutation (E14Q) was engineered into the EmrEⁱⁿ or EmrE^out constructs (i.e. E14Qⁱⁿ or E14Q^out). E14Q was selected since EmrE requires two glutamic acid residues to confer drug resistance (Rapp et al., 2007). Thus, if a dimer of wild-type and E14Qⁱⁿ or E14Q^out formed, we would observe a reduced growth phenotype. Indeed, the results of these experiments displayed growth inhibition when wild-type was co-expressed with E14Qⁱⁿ or E14Q^out (Figure 5A). Taken together with the robust phenotype of wild-type co-expressed with EmrEⁱⁿ or EmrE^out, these results confirmed that the transporter complementation approach leads to heterodimer formation in the cell membrane.

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We next carried out experiments with L51I and I62L co-expressed with EmrEⁱⁿ or EmrE^out. Remarkably, when EmrEⁱⁿ was co-expressed with L51I or I62L, we observed a significant reduction in ethidium resistance on LB agar plates (Figure 5B). To the contrary, EmrE^out co-expressed with L51I or I62L retained the same phenotype as the single-site mutant alone. To provide additional support, we carried out resistance assays in liquid media. These experiments were performed by growing E. coli cultures containing plasmids to an optical density at 600 nm of 1.0, then adding ethidium and recording the culture density as a function of time. Similar to the LB agar plate results, we found that the L51I or I62L mutant expressed with EmrEⁱⁿ displayed a faster drop in the optical density, which signified a reduced ability to confer resistance relative to the same mutant expressed with EmrE^out (Figure 5C). It is important to note that if heterodimers of L51I or I62L and EmrEⁱⁿ did not form, we would have observed the same resistance phenotype of L51I or I62L alone. However, the reduced phenotype and the control experiments discussed above (Figure 5A; Figure 5—figure supplement 1) indicate a specific association in the transporter complementation experiments that are influenced by the conformational equilibrium within the assembled heterodimer in the cell membrane.

To provide direct evidence that the overall rate of ethidium transport was different between the mutant co-expressed with EmrEⁱⁿ and EmrE^out, we performed an ethidium efflux assay by measuring the intrinsic fluorescence of ethidium. E. coli were treated with ethidium bromide and the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which causes the cytoplasmic ethidium concentration and fluorescence to increase. Upon addition of glucose and removal of CCCP, the membrane potential is reestablished, and the fluorescence decreases as ethidium is transported out of the cytoplasm. This assay was performed with L51I co-expressed with EmrEⁱⁿ or EmrE^out. Immediately following the addition of glucose, the fluorescence dropped ~3 fold faster for L51I co-expressed with EmrE^out than L51I co-expressed with EmrEⁱⁿ (Figure 5D). Furthermore, the fluorescence value at steady state (3600 s) was significantly lower for L51I co-expressed with EmrE^out compared to L51I co-expressed with EmrEⁱⁿ (Figure 5E). This indicates a reduced cytoplasmic ethidium concentration for the L51I/EmrE^out sample. Taken together, these data are consistent with the resistance assay results and support the conclusion that a change in the conformational equilibrium can influence the overall rate of drug efflux. Note that a previous study reported an ethidium efflux rate difference of 1.6-fold between wild-type E. coli and an AcrAB-TolC efflux pump knockout strain that led to a 30-fold difference in the minimum inhibitory concentration (Paixão et al., 2009). This shows that somewhat modest transport rate differences can have deleterious effects on bacterial growth, which stems from increased ethidium accumulation that leads to irreversible binding to DNA (Jernaes and Steen, 1994; Lambert and Le Pecq, 1984).

How does the NMR observed conformational change of the heterodimer correlate with the functional results? The transporter complementation experiments create topologically defined heterodimers due to the single topology of EmrEⁱⁿ and EmrE^out and the antiparallel association of the dimer quaternary structure. Using this information and knowledge from NMR experiments that the mutant had a preference for monomer A in the heterodimer, we can make inferences about how a shift in the conformational equilibrium influences function. Specifically, the mutant paired with EmrE^out in a heterodimer would favor the inward-open/cytoplasmic-facing conformation (Figure 5F). Likewise, the mutant paired with EmrEⁱⁿ will bias toward the outward-open/periplasmic-facing conformation under the same conditions (Figure 5F). Hence, the resistance assay results suggest that the conformational equilibrium favoring the outward-open/periplasmic state has a deleterious impact on the overall transport cycle. In contrast, a heterodimer with a conformation favoring the inward-open/cytoplasmic facing conformation gives no measurable reduction in phenotype. This finding is in harmony with our prior observation that the role of the pH gradient is to favor the inward-open conformation that is poised for drug binding (Gayen et al., 2016). Thus, reducing the effect of the pH gradient by biasing the equilibrium in the opposite direction apparently is sufficient to alter the net transport as observed in growth inhibition experiments and efflux assays.

It is important to underscore that the equilibrium constant between inward-open and outward-open states of the heterodimer ultimately stems from differential kinetic rate constants pertaining to the inward-open to outward-open and outward-open to inward-open transitions (see kinetic model [Robinson et al., 2017] in Figure 5G). Thus, we hypothesize that the primary kinetic steps in the transport cycle that influence the observed phenotype are the inward-open to outward-open and outward-open to inward-open rates for the drug-free heterodimer (Figure 5G, see double asterisks). We make this conclusion based on two observations. First, the populations were more skewed for the drug-free states than those for the drug bound states. Second, NMR experiments with ethidium indicate that the conformational exchange was not halted in the heterodimer. The latter means that the conformational bias does not result in a locked-state that would ultimately lead to loss-of-function (i.e. no substrate turnover).

To provide support for this conclusion, we performed a numerical simulation of pH-driven transport into liposomes using the kinetic model and parameters introduced by Robinson et al. (2017). The differences were to change the inward-open to outward-open and outward-open to inward-open conformational exchange rates based on the populations observed in our heterodimer experiments and to account for the fact that drug binding affinities to wild-type(A)/mutant(B) and mutant(A)/wild-type(B) are not necessarily the same (see Figure 5—figure supplement 2). Using these simulations, we found that the rate of drug transport was ~5.6 fold faster for a heterodimer of mutant and EmrE^out versus a heterodimer of mutant and EmrEⁱⁿ (Figure 5—figure supplement 2). In addition, the former achieved a small but significant increase in the final concentration of drug within the vesicles, which further suggests that differences in kinetic rates in EmrE’s transport cycle give rise to a change in the cytoplasmic drug concentration between heterodimers with opposite insertion topologies in E. coli. These calculations correlate with the enhanced phenotype and increased rate of ethidium efflux and provide evidence that shifting conformational equilibria via specific rate constants in a transport cycle has a direct impact on functional output.

Despite the prominence of inverted repeat domains within transporters, there are only a few examples of mutational studies to influence conformational equilibria. These experiments involve the creation of loss-of-function, non-conservative mutations (e.g. Trp to Gly) at key structural contacts with the goal of crystallographically trapping particular conformations in the transport cycle (Smirnova et al., 2013; Latorraca et al., 2017). Our observations demonstrate that a minimal heterodimer comprised of a single conservative mutation is sufficient to disrupt the preferred resting conformation and underscore the presence of a dynamic energy landscape where relatively small energy differences exist among conformations in the transport cycle. From an evolutionary perspective, we hypothesize there might be fitness advantages for having separate genes that form a hetero-oligomer or a single polypeptide chain where the structural repeats are contained within one protein. In these cases, a single mutation can be introduced within the functional assembly that is not possible for a homo-oligomeric protein. Functional differences between closely related proteins stemming from one mutation in a hetero-oligomer versus multiple mutations in a homo-oligomer could potentially include influencing the propensity of a transporter to carry out antiport or symport. In fact, in addition to EmrE’s known antiport activity, it is also able to carry out symport under certain conditions (Robinson et al., 2017), including import of polyamines upon mutation (Brill et al., 2012). Therefore, the observation that conservative mutations can display skewed conformational equilibria and influence function opens the possibility of somewhat modest evolutionary paths for achieving symport or antiport.

Protein expression and purification was carried out as previously reported (Gayen et al., 2013). EmrE is expressed as a fusion construct with maltose-binding protein in the pMAL vector (New England Biolabs Inc) in E. coli BL21(DE3) cells. For oriented sample NMR experiments, ¹⁵N-tyrosine labeled EmrE was expressed with IPTG for 4 hr at 25°C in an amino acid mixture consisting of 19 amino acids at natural abundance (300 mg/L) and ¹⁵N-tyrosine (120 mg/L). Unlabeled EmrE used in the heterodimer experiments was expressed in LB medium at natural abundance. For solution NMR experiments, wild-type and L51I were expressed in a fully perdeuterated background as previously described (Gayen et al., 2016). Wild-type protein was isotopically enriched with ¹³C at the C^δ position of isoleucine methyl groups with the addition of 50 mg/L 2-ketobutyric acid-4-¹³C, 3,3-²H₂ sodium salt hydrate 1 hr before induction. The bacterial cells following expression were lysed and the fusion protein was purified with amylose affinity chromatography. The fusion protein was cleaved with tobacco etch virus protease (TEV) and EmrE was further purified by size exclusion chromatography using a Superdex 200 10/300 column (GE Healthcare) in 0.06% w/v n-dodecyl-β-D-maltopyranoside (DDM).

For oriented sample solid-state NMR studies, purified EmrE in DDM detergent was reconstituted into 1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine/dihexanoyl-sn-glycero-3-phosphocholine (O-14:0-PC/6:0-PC) bicelles at a molar ratio of 3.5/1 (i.e. q = 3.5). The bicelles were made with a protein concentration ~2 mM with a combined lipid concentration of 25% (w/v) in 80 mM HEPES and 20 mM NaCl at pH = 5.4. The heterodimer samples were prepared by mixing the two proteins (wild-type/L51I or wild-type/I62L) with a molar ratio 1/1.2, where the protein in excess was at natural abundance (i.e. NMR silent). The two proteins used to prepare the heterodimer sample were incubated together at 37°C in the presence of 50 mM DTT for 1 hr immediately prior to reconstitution into lipids. The reconstitution into the bicelles was carried out in the same manner as those for homodimer samples (Leninger and Traaseth, 2018).

For solution NMR studies, purified EmrE in DDM detergent was reconstituted into dimyristoyl-sn-glycero-3-phosphocholine (14:0-PC) and dihexanoyl-sn-glycero-3-phosphocholine (6:0-PC) bicelles with a molar ratio of 1/2. The acyl chains of 14:0-PC and 6:0-PC were perdeuterated (Avanti Polar Lipids) to reduce the lipid signals in ¹H/¹³C heteronuclear correlation experiments. The heterodimer samples were prepared by mixing isotopically enriched wild-type with natural abundance L51I in a 1/3 molar ratio. The two proteins used to prepare the heterodimer sample were incubated together at 37°C for 1 hr immediately prior to reconstitution into lipids. The heterodimers contained 0.533 mM total protein (0.133 mM wild-type, 0.4 mM L51I). The long-chain lipid (14:0-PC) to total protein ratio of the solution NMR samples was ~150/1 (mol/mol). Control homodimer samples with isotopically enriched wild-type EmrE only were prepared in a similar fashion. The sample buffer was 150 mM Na₂HPO₄ and 20 mM NaCl. The experiments with ethidium bromide and tetraphenylphosphonium were carried out at concentrations of 2.1 mM and 1.05 mM, respectively.

In order to assess whether a 1 hr incubation time at 37°C was sufficient to achieve complete mixing of the heterodimer, a control experiment was carried out by comparing NMR spectra after 1 hr and 19 hr of incubation at 37°C. The NMR spectra collected of these experiments showed that shorter and longer incubation times gave statistically the same ratios of monomer A and B peak intensities, which demonstrates that complete mixing was achieved with 1 hr (Figure 3—figure supplement 1B). Note that the 1 hr incubation time in DDM is consistent with the reduced dimer stability of EmrE in DDM detergent micelles compared to that in lipid bicelles or lipid bilayers (Dutta et al., 2014a).

Oriented sample solid-state NMR experiments were acquired using an Agilent DD2 spectrometer at a ¹H frequency of 600 MHz equipped with a ¹H/¹⁵N double resonance probe manufactured by Revolution NMR (design of Peter Gor’kov; Gor'kov et al., 2007). Experiments were carried out using magnetically aligned lipid bicelle samples that were flipped with the addition of 3 mM YbCl₃ to orient the bicelle normal parallel to the magnetic field. PISEMA (Wu et al., 1994) spectra were acquired with SPINAL-64 ¹H decoupling during acquisition and phase modulated Lee-Goldberg (Vinogradov et al., 1999) (PMLG) ¹H-¹H decoupling in the indirect dimension. The radiofrequency field for SPINAL decoupling was 50 kHz, while the effective field for PMLG was 41.7 kHz. Spectra were acquired with ~1500 scans and 14 increments in the indirect dimension with a recycle delay of 3 s. The indirect dimension axis was corrected with the scaling factor of 0.82. The ¹⁵N direct dimension was referenced to ¹⁵NH₄Cl (solid) at 41.5 ppm.

Solution NMR experiments were acquired using a Bruker Avance III spectrometer at a ¹H frequency of 600 MHz equipped with a TCI cryogenic probe. ¹H/¹³C HMQC experiments were acquired at 25°C using ¹H and ¹³C spectral widths of 10,000 Hz and 4,000 Hz, respectively. The total acquisition (¹H) and evolution times (¹³C) corresponding to t₂ and t₁ were 59.9 msec and 18.5 msec, respectively. Spectra were acquired with 12 or 24 scans with a recycle delay of 1 s.

The equilibrium shown in Equation 1 corresponds to a heterodimer composed of wild-type EmrE and mutant:

Subscripts 'A' and 'B' correspond to monomers A and B in the asymmetric dimer. A simple calculation of the peak intensity ratio for A and B resonances in the heterodimer samples does not correspond to the equilibrium constant since the peak intensities contain a statistical fraction of homodimers ($f_{homo}$) and heterodimers ($f_{het}$) based on the ratio of isotopically labeled and natural abundance proteins present for mixing. Note that $f_{homo}$ and $f_{het}$ consider only homodimers or heterodimers that contain the isotopically enriched protein. The observed peak intensities for monomer A ($I_{A,obs}$) and B ($I_{B,obs}$) signals in a heterodimer sample are given by Eqns. 2 and 3:

Equation 2 divided by Equation 3 gives:

$I_A$/$I_B$ is the ratio of ‘intrinsic’ intensities of monomer A and B peaks obtained from the homodimer spectrum. This is needed since A and B peaks in the homodimer spectrum are not exactly the same. $f_{homo}$ and $f_{het}$ were set to 1/7 and 6/7, respectively, by assuming statistical mixing of the molar ratio of isotopically labeled wild-type to natural abundance L51I mutant in solution NMR experiments (i.e., 1 part labeled to 3 parts unlabeled). $p_A$ and $p_B$ are populations of wild-type in monomer A or B conformations when in a heterodimer with a mutant (see Equation 1). The addition of these populations is given by Equation 5 and their ratio is the equilibrium constant (K) in Equation 6:

Substitution of $p_B$ from Equation 5 into Equation 4 gives the following:

$p_A$ was solved from Equation 7 for drug-free (pH = 9.1) and tetraphenylphosphonium bound forms of wild-type/L51I heterodimers using solution NMR HMQC spectra of wild-type EmrE and isotopically labeled wild-type mixed with natural abundance L51I. The average and error range (derived from the standard deviation) of the populations were determined from the following residues that displayed well-resolved signals in each spectrum: Ile11, Ile54, Ile58, Ile62, Ile68, Ile88, and Ile101.

Growth inhibition assays were performed in pET Duet-1 vectors with constructs designed similar to that of Rapp et al. (2007). All of the constructs are shown in Figure 4—figure supplement 1. EmrEⁱⁿ consists of three mutations relative to wild-type EmrE (R29G, R82G, S107K) and induces the N- and C-termini to face the cytoplasm. EmrE^out consists of three mutations from wild-type (T28R, L85R, R106A) and induces the N- and C-termini to face the periplasm. EmrEⁱⁿ and EmrE^out were placed in the second cloning site. An additional mutation to EmrEⁱⁿ and EmrE^out constructs were made by mutating E14 to Q14. These constructs are also single topology and are referred to as E14Qⁱⁿ and E14Q^out. The L51I and I62L constructs are single site mutants of wild-type EmrE (UniProt P23895).

Luria-Bertani (LB) media and Luria agar powder for resistance assay were purchased from Research Products International. Each construct in the pET Duet-1 vector was transformed into BL21(DE3) and grown at 37°C up to an OD₆₀₀ of ~1.5. The cultures were diluted to an OD₆₀₀ of 1.0 with fresh LB supplemented with carbenicillin (100 μg/mL) and were then serially diluted by 10-fold to achieve final dilutions of 10⁰ to 10⁶. All of the dilutions used fresh LB media containing 100 μg/mL carbenicillin. 3 μl of each dilution were pipetted onto plates containing 20 μM IPTG, 100 μg/mL carbenicillin, and 94 μg/ml ethidium bromide. Control experiments were carried out by plating cells onto LB agar plates containing 20 μM IPTG and 100 μg/mL carbenicillin. All resistance assays on solid medium were repeated at least two times.

Liquid assays were performed using the same constructs as for the solid media resistance assay. E. coli BL21 (DE3) were transformed with the pET Duet-1 vectors and grown at 37°C until the OD₆₀₀ was ~1.5. The cultures were then diluted to an OD₆₀₀ of 1.0 with fresh LB supplemented with carbenicillin (100 μg/mL) and ethidium bromide (240 μg/mL). Cultures were incubated for 11.5 hr and the OD₆₀₀ was measured every hr up to 6 hr and one additional time 11.5 hr after the initial exposure to ethidium bromide. A full set of resistance assays in liquid medium were repeated three times. The error bars reflect the standard deviation for replicate trials.

Mutant constructs used in resistance assay were transformed into BL21(DE3) and grown to mid log phase at 37°C (OD₆₀₀ ~1.0). The cells were spun down and diluted to the OD₆₀₀ of 0.1 in minimal media A (40 mM K₂HPO₄, 22 mM KH₂PO₄, 2 mM sodium citrate, 0.8 mM MgSO₄, and 7.6 mM (NH₄)₂SO₄, pH 7.0). The resuspended cells were treated with 80 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for 5 min. Ethidium bromide (10 μg/mL) was added to the cells and incubated for 30 min at 37°C while shaking. Cells were spun down for 10 min and the pellet was kept on ice until the florescence experiment. For the efflux assay, cells were resuspended in minimal media A with 10 μg/mL ethidium bromide. To initiate the assay, 0.2% (w/v) glucose was added. Control experiments were carried out by treating the cells in the same manner except without adding glucose. The fluorescence decays were measured with a Molecular Devices FlexStation 3 instrument using an excitation wavelength of 530 nm and an emission wavelength of 600 nm. The fluorescence was recorded over a time period of 3600 s. Each experiment was acquired in duplicate and repeated at least two times. The error bars reflect the duplicate experiments carried out on the same day.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This work was supported by NIH (R01 AI108889) and NSF awards (MCB 1506420) to NJT. ML was supported from a Dean’s Dissertation Fellowship from New York University. The NMR data collected with a cryoprobe at NYU were supported by an NIH S10 grant (OD016343).

© 2019, Leninger et al.

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