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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

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.
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fig4: 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.

Mentions: Second, we can perform a pH titration of a Glu14 mutant to confirm that we are specifically detecting protonation of Glu14 (Figs. 4 and S2). If the hypothesis that we are observing a global structural change upon Glu14 protonation is true, then (a) all residues should sense a single identical pKa, and (b) mutation of Glu14 to another residue should change the pH dependence of all residues in an identical manner. If the alternative is true and the NMR experiments detect only local effects of many separate protonation events at individual Glu, Asp, and His residues, then (a) different pKa values should be observed for each local region, and (b) mutation of Glu14 should only cause pH-dependent chemical-shift changes for residues in close proximity. To test the effect of mutation of Glu14, we chose E14D-EmrE because it is the only E14 mutation that retains any function (Yerushalmi and Schuldiner, 2000b). All other mutations of Glu14 render EmrE unable to transport or even bind substrate, so it is more difficult to assess that the protein is properly folded and otherwise well behaved. Based on previous biochemical estimates, the pKa of the active site is shifted from ≈7.3 in WT-EmrE to ≈5.4 in E14D-EmrE (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b), a significant shift that is easily detectable in our titrations. This is exactly what is observed in the spectra (Figs. 4 and S2). The same residues in EmrE still titrate, but the pH-dependent chemical-shift changes occur at more acidic pH, exactly as expected. These results show that the NMR titrations are monitoring the protonation state of Glu14 coupled to a global conformational change. Quantitative analysis of all of the titrations is discussed below.


Asymmetric protonation of EmrE.

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

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

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

fig4: 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.
Mentions: Second, we can perform a pH titration of a Glu14 mutant to confirm that we are specifically detecting protonation of Glu14 (Figs. 4 and S2). If the hypothesis that we are observing a global structural change upon Glu14 protonation is true, then (a) all residues should sense a single identical pKa, and (b) mutation of Glu14 to another residue should change the pH dependence of all residues in an identical manner. If the alternative is true and the NMR experiments detect only local effects of many separate protonation events at individual Glu, Asp, and His residues, then (a) different pKa values should be observed for each local region, and (b) mutation of Glu14 should only cause pH-dependent chemical-shift changes for residues in close proximity. To test the effect of mutation of Glu14, we chose E14D-EmrE because it is the only E14 mutation that retains any function (Yerushalmi and Schuldiner, 2000b). All other mutations of Glu14 render EmrE unable to transport or even bind substrate, so it is more difficult to assess that the protein is properly folded and otherwise well behaved. Based on previous biochemical estimates, the pKa of the active site is shifted from ≈7.3 in WT-EmrE to ≈5.4 in E14D-EmrE (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b), a significant shift that is easily detectable in our titrations. This is exactly what is observed in the spectra (Figs. 4 and S2). The same residues in EmrE still titrate, but the pH-dependent chemical-shift changes occur at more acidic pH, exactly as expected. These results show that the NMR titrations are monitoring the protonation state of Glu14 coupled to a global conformational change. Quantitative analysis of all of the titrations is discussed below.

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