Asymmetric protonation of EmrE

Abstract

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its "active-site" residues--glutamate 14 (Glu14) from each subunit--must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with (1)H-(15)N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.

Figure 1.

The mechanism of EmrE transport. EmrE is a homodimer composed of two subunits (blue and pink) oriented antiparallel to each other and in unique conformations (distinct shapes, labeled A and B). The subunits exchange conformations to switch between open-in and open-out forms. (A) In the standard single-site alternating access model, a single active site located between the two subunits alternates between binding protons or drug. Coupling between protons and drug is achieved by preventing exchange in the apo state and only allowing one substrate to bind at a time. (B) Drug-free EmrE has more states than represented in the basic model shown in A and most, if not all, of these states exchange.

Figure 2.

Structure of homodimeric TPP⁺-bound EmrE with pH-sensitive residues highlighted. Basic residues are colored blue, and acidic residues are colored red. TPP⁺ is not shown. Note that Glu14, shown as sticks with the protonatable oxygen represented as a ball, is the only charged residue in the TM regions. Yellow residues (Ala10, Gly17, and Ser43) were used for the pKa fits and are located near the Glu14 active site. Black residues are more remote from the active site but also fit to the same two pKa values determined using the yellow residues close to Glu14 (Protein Data Bank accession no. 3B5D).

Figure 3.

Drug-free EmrE is sensitive to pH. Full ¹H-¹⁵N TROSY-HSQC spectra of drug-free WT EmrE in DLPC/DHPC isotropic bicelles at three pH values collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. All spectra in the full pH titration datasets are shown in Figs. S1 (45°C) and S3 (25°C). Circles highlight several residues that remain in the slow exchange regime at 25°C at low pH.

Figure 4.

E14D-EmrE has a shifted pH sensitivity. ¹H-¹⁵N TROSY-HSQC spectra of drug-free E14D-EmrE in DMPC/DHPC isotropic bicelles at 45°C and varying pH values. Note that chemical shifts of His110 and Ser105 (circled peaks) titrate with a higher pKa than the rest of the protein. The pH of each spectrum is indicated by its color as designated in the figure. The full set of pH titration spectra are shown in Fig. S2.

Figure 5.

Ala10 senses the protonation state of EmrE. Enlarged region of the ¹H-¹⁵N TROSY-HSQC spectra showing the Ala10 peaks for both WT EmrE (A and B) and E14D-EmrE (C). Spectra were collected at 45°C (A and C) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of peaks as the pH is lowered. When two distinct subunit peaks can be distinguished, the assignment of each subunit is labeled as A and B. (B) Exchange cross peaks are marked with an “x.” (C) The four peaks at pH 5.0 (green spectrum) are interpreted as two states of the E14D-EmrE dimer, indicated with ○ and *. These peaks do not reflect conformational exchange because they do not align in a square pattern. Both subunits have a distinct conformation in each state. The dashed line highlights the additional slow-exchange process at low pH.

Figure 6.

Gly17 senses the protonation state of EmrE. Enlarged glycine region of the ¹H-¹⁵N TROSY-HSQC spectrum for WT EmrE. Spectra were collected at 45°C (A) and 25°C (B). The pH of each spectrum is indicated by its color as designated in the figure. Lines highlight the movement of the peaks as the pH is lowered.

Figure 7.

Simulation of different NMR titration patterns. (A) For a single residue, each unique conformation or state of a protein will have a distinct chemical shift. When these conformations or states are able to exchange, the pattern observed in the NMR spectrum depends on the rate of the exchange process (k_ex) relative to the frequency difference between the chemical shifts of the two species (Δω). In A, one-dimensional NMR spectra are simulated for Δω = 100 Hz. When k_ex is much slower than Δω (bottom, slow-exchange regime), two peaks are observed at the unique chemical shifts of these two conformations or states. The area under each peak reflects the relative population of each, 25%/75% in this example. As the exchange rate increases, the peaks broaden and merge, eventually resulting in a single narrow peak at the population-weighted average chemical shift of the exchanging species (top, fast-exchange regime). NMR spectra are usually reported with axes in units of parts per million (ppm) to remove their dependence on the spectrometer field strength. However, the chemical shift actually corresponds to a frequency. For the spectra presented here, acquired on a 700-MHz NMR spectrometer, 1 ppm in proton corresponds to 700 Hz. (B) In the fast-exchange regime, where k_ex is fast compared with Δω, titration results in a shift in peak position, from the free to bound state as ligand is added and the relative population of the free and bound states changes. (C) In the slow-exchange regime, the free state disappears and the bound state appears during the course of a titration as ligand is added and the population shifts from free to bound. Intermediate exchange will result in a combination of peak shifting and broadening. Because proton on/off is generally fast, we expect (and observe) spectra where peaks shift position with pH. (D) In the case of two-state exchange from a protonated state, marked H, to a deprotonated state, marked D, the peak position in a two-dimensional spectrum will move along a line connecting the peaks corresponding to the fully protonated and fully deprotonated states as pH is changed. (E) Plotting the position of the peak (in ppm) as a function of pH will result in a classical binding curve, reflecting the nonlinear dependence of the fraction protonated on pH. Thus, the chemical shift can be analyzed in the same way as any other protein property that is sensitive to protonation-state changes. The exact equations are given in Materials and methods. (F) If the protein is exchanging between three states (2H, two protons bound; 1H, one proton bound; D, deprotonated, no protons bound), each with unique chemical shifts, then the peak position will reflect the population-weighted average of all three chemical shifts at each pH value. An example is shown for two protonation steps assuming non-interacting sites with pKa values separated by 1.4 pH units. The averaging of three states results in a curved path of the peak across the spectrum. (G) The fraction doubly protonated (solid line), singly protonated (dotted), and deprotonated (dashed) are shown along with the peak position (in parts per million) as a function of pH. Both transitions are clearly observed in the peak position, although the chemical-shift difference between states 1H and D is smaller along the proton dimension. Eq. 2 in Materials and methods takes into account the relative chemical shifts of all three states.

Figure 8.

The pH-dependent chemical shifts of drug-free WT EmrE at 45°C fit to two pKa values. Proton (A and B) and nitrogen (C and D) chemical shifts were plotted independently as a function of pH for both backbone amides (A and C) and tryptophan side chains (sc) (B and D). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data for residues marked with closed symbols were fit to both a single pKa model (dotted lines; pKa = 7.1 ± 0.1; n = 0.7) and a double pKa model (solid lines; pKa = 7.0 ± 0.1 and 8.2 ± 0.3). Open symbols indicate residues that were not used to determine the pKa values but can be fit to the same pKa values.

Figure 9.

The pH-dependent chemical shifts of drug-free WT EmrE at 25°C fit to two pKa values, similar to those at 45°C. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH for both backbone amides and tryptophan side chains (sc). Each y axis is labeled with the residues it represents. The residues are indicated by color as displayed in the figure. The data were globally fit to both a single pKa model (dotted lines; pKa = 6.9 ± 0.1; n = 0.8) and a double pKa model (solid lines; pKa = 6.8 ± 0.1 and 8.5 ± 0.2).

Figure 10.

The pH-dependent chemical shifts of Ala10 from drug-free E14D-EmrE fit to two significantly shifted pKa values. Proton (A) and nitrogen (B) chemical shifts were plotted independently as a function of pH. Blue and green symbols represent subunit A, and black and red symbols represent subunit B. Near the pKa, each subunit splits into a major and minor peak resulting in the multiple colors to represent the two states of each subunit. The data were globally fit to a double pKa model (solid lines; pKa = 5.2 ± 0.1 and 5.5 ± 0.1). The same pKa values are obtained if the major and minor states are independently fit to single pKa values.

Figure 11.

Drug-free EmrE exchanges at high pH. Representative cross peaks (connected to auto peaks by blue squares) for well-resolved residues. (A) Cross peaks are visible in the ¹H-¹⁵N TROSY-HSQC of WT EmrE in isotropic bicelles at pH 8.4 collected at 45°C (red) as compared with the ¹H-¹⁵N TROSY-HSQC collected at 25°C (navy). (B and C) TROSY-selected ZZ-exchange experiments show cross peaks at lower temperatures. (B) Regions of a ZZ-exchange spectrum collected on WT EmrE in isotropic bicelles at pH 8.4, 35°C, with a 50-ms mixing time (orange) overlaid with a matching ¹H-¹⁵N TROSY-HSQC (black). (C) Regions of a ZZ-exchange spectrum collected on WT EmrE in DMPC/DHPC isotropic bicelles at pH 8.2, 25°C, with an 80-ms mixing time (cyan) overlaid with a matching ¹H-¹⁵N TROSY-HSQC (navy).