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Asymmetric protonation of EmrE.

Morrison EA, Robinson AE, Liu Y, Henzler-Wildman KA - J. Gen. Physiol. (2015)

Bottom Line: 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.

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Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus

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 (kex) 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 kex 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 kex 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.
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fig7: 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 (kex) 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 kex 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 kex 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.

Mentions: As the pH is lowered at 45°C, the two peaks corresponding to subunit A and subunit B merge into a single peak for many residues, such as Ala10 (Figs. 3 A, 5 A, and 6 A). A single peak for each residue would be observed if either (a) the exchange between conformations increases and becomes fast on the NMR timescale (i.e., the exchange rate is significantly larger than the chemical-shift difference; see Fig. 7 A), or (b) asymmetry is lost and the two subunits have identical conformations. We can differentiate between these two options by looking at spectra collected at a lower temperature (25°C; Figs. 3 B, 5 B, and 6 B), which decreases the rate of conformational exchange. Some residues such as Gly26 or Gly80 still have two distinct peaks visible at low pH at 25°C (Fig. 3 B). The reason that some residues have a single peak at low pH (such as Ala10; Fig. 5 B) and some have two peaks at low pH in this 25°C spectrum is because of the definition of the NMR “timescale” (Fig. 7 A), which depends on the exchange rate relative to the chemical-shift difference. Although there is a single global conformational exchange process (subunits A and B swapping conformations to convert from the open-in to open-out state) with a single global rate, the chemical-shift difference between the subunit A and subunit B peaks is a residue-specific property. Some residues in EmrE exist in very similar environments in subunits A and B, whereas other residues have very different environments in the two subunits and thus large chemical-shift differences (see Morrison et al., 2012, for further discussion of the chemical-shift differences between the two subunits in the asymmetric EmrE homodimer). For the same global exchange rate, some residues with large chemical-shift differences will fall in the slow exchange regime, and two peaks will be observed even at low pH, as for Gly26 and Gly80 at 25°C (Fig. 3 B). Residues with smaller chemical-shift differences will fall in the fast exchange regime, and only a single peak will be observed at low pH, as for Ala10 (Fig. 5 B). Even for residues in the slow exchange regime, the two peaks visible at low pH are broader and weaker than the two peaks at high pH for the same residue, reflecting an increased exchange rate. Thus, the data appear to report the behavior predicted by the single-site alternating access model for coupled antiport: EmrE should interconvert rapidly when bound to substrate (protons) and should not exchange in the absence of substrate (high pH, deprotonated). However, closer inspection of the spectra reveals greater complexity.


Asymmetric protonation of EmrE.

Morrison EA, Robinson AE, Liu Y, Henzler-Wildman KA - J. Gen. Physiol. (2015)

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 (kex) 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 kex 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 kex 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.
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fig7: 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 (kex) 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 kex 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 kex 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.
Mentions: As the pH is lowered at 45°C, the two peaks corresponding to subunit A and subunit B merge into a single peak for many residues, such as Ala10 (Figs. 3 A, 5 A, and 6 A). A single peak for each residue would be observed if either (a) the exchange between conformations increases and becomes fast on the NMR timescale (i.e., the exchange rate is significantly larger than the chemical-shift difference; see Fig. 7 A), or (b) asymmetry is lost and the two subunits have identical conformations. We can differentiate between these two options by looking at spectra collected at a lower temperature (25°C; Figs. 3 B, 5 B, and 6 B), which decreases the rate of conformational exchange. Some residues such as Gly26 or Gly80 still have two distinct peaks visible at low pH at 25°C (Fig. 3 B). The reason that some residues have a single peak at low pH (such as Ala10; Fig. 5 B) and some have two peaks at low pH in this 25°C spectrum is because of the definition of the NMR “timescale” (Fig. 7 A), which depends on the exchange rate relative to the chemical-shift difference. Although there is a single global conformational exchange process (subunits A and B swapping conformations to convert from the open-in to open-out state) with a single global rate, the chemical-shift difference between the subunit A and subunit B peaks is a residue-specific property. Some residues in EmrE exist in very similar environments in subunits A and B, whereas other residues have very different environments in the two subunits and thus large chemical-shift differences (see Morrison et al., 2012, for further discussion of the chemical-shift differences between the two subunits in the asymmetric EmrE homodimer). For the same global exchange rate, some residues with large chemical-shift differences will fall in the slow exchange regime, and two peaks will be observed even at low pH, as for Gly26 and Gly80 at 25°C (Fig. 3 B). Residues with smaller chemical-shift differences will fall in the fast exchange regime, and only a single peak will be observed at low pH, as for Ala10 (Fig. 5 B). Even for residues in the slow exchange regime, the two peaks visible at low pH are broader and weaker than the two peaks at high pH for the same residue, reflecting an increased exchange rate. Thus, the data appear to report the behavior predicted by the single-site alternating access model for coupled antiport: EmrE should interconvert rapidly when bound to substrate (protons) and should not exchange in the absence of substrate (high pH, deprotonated). However, closer inspection of the spectra reveals greater complexity.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.

No MeSH data available.


Related in: MedlinePlus