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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.

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

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.
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fig10: 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.

Mentions: Close examination of the E14D pH titration (Figs. 4 and 5 C) reveals a significant difference in the relative timescales of the protonation and conformational exchange processes compared with WT EmrE. It is easiest to see the pattern in the Ala10 peaks. At high pH, two peaks corresponding to the two subunits in the homodimer are visible as in WT. This indicates that conformational exchange between open-in and open-out E14D-EmrE is slow at high pH (deprotonated EmrE). As the pH decreases, the peaks move along a trajectory (Fig. 5 C, solid arrows), as expected for fast proton on/off, analogous to WT EmrE. However, at pH 5, near the pKa reported from binding assays, four distinct peaks are visible (Fig. 5 C, green spectrum, pH 5) as two pairs of peaks (*) and (○), with each pair having relatively equal intensity. One peak from each pair lies along the titration path for subunit A, and one peak from each pair lies along the titration path for subunit B. This suggests that there may be two states of the EmrE dimer (* and ○) resulting in two conformations of subunit A and two conformations of subunit B. Based on the relative peak intensities, these two states of the dimer have unequal populations. Given this understanding of the unique pattern of peak movement, the chemical shifts of Ala10 as a function of pH can be fit to two pKa values of 5.2 ± 0.1 and 5.5 ± 0.1 for the major and minor peaks, respectively (Fig. 10).


Asymmetric protonation of EmrE.

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

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4664823&req=5

fig10: 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.
Mentions: Close examination of the E14D pH titration (Figs. 4 and 5 C) reveals a significant difference in the relative timescales of the protonation and conformational exchange processes compared with WT EmrE. It is easiest to see the pattern in the Ala10 peaks. At high pH, two peaks corresponding to the two subunits in the homodimer are visible as in WT. This indicates that conformational exchange between open-in and open-out E14D-EmrE is slow at high pH (deprotonated EmrE). As the pH decreases, the peaks move along a trajectory (Fig. 5 C, solid arrows), as expected for fast proton on/off, analogous to WT EmrE. However, at pH 5, near the pKa reported from binding assays, four distinct peaks are visible (Fig. 5 C, green spectrum, pH 5) as two pairs of peaks (*) and (○), with each pair having relatively equal intensity. One peak from each pair lies along the titration path for subunit A, and one peak from each pair lies along the titration path for subunit B. This suggests that there may be two states of the EmrE dimer (* and ○) resulting in two conformations of subunit A and two conformations of subunit B. Based on the relative peak intensities, these two states of the dimer have unequal populations. Given this understanding of the unique pattern of peak movement, the chemical shifts of Ala10 as a function of pH can be fit to two pKa values of 5.2 ± 0.1 and 5.5 ± 0.1 for the major and minor peaks, respectively (Fig. 10).

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