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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

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.
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fig5: 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.

Mentions: Closer inspection of the titration patterns of individual residues, particularly Ala10 (Fig. 5) and the glycine region (Fig. 6), reveals that the conformational exchange rate between open-in and open-out states of EmrE is also pH dependent. At high pH, two peaks are observed for each residue of EmrE at 25 and 45°C (Fig. 3). The two peaks present in the spectra of drug-free EmrE at high pH have very similar chemical shifts to the drug-bound spectra of EmrE that we have analyzed previously (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014) and can be assigned to subunit A and subunit B of the asymmetric homodimer. We have shown previously that the two subunits swap conformations as drug-bound EmrE converts from open-in to open-out (Morrison et al., 2012). Whether the two subunits swap conformations at high pH for drug-free EmrE is discussed below in the section, Is apo EmrE still able to interconvert?


Asymmetric protonation of EmrE.

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

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

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

fig5: 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.
Mentions: Closer inspection of the titration patterns of individual residues, particularly Ala10 (Fig. 5) and the glycine region (Fig. 6), reveals that the conformational exchange rate between open-in and open-out states of EmrE is also pH dependent. At high pH, two peaks are observed for each residue of EmrE at 25 and 45°C (Fig. 3). The two peaks present in the spectra of drug-free EmrE at high pH have very similar chemical shifts to the drug-bound spectra of EmrE that we have analyzed previously (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014) and can be assigned to subunit A and subunit B of the asymmetric homodimer. We have shown previously that the two subunits swap conformations as drug-bound EmrE converts from open-in to open-out (Morrison et al., 2012). Whether the two subunits swap conformations at high pH for drug-free EmrE is discussed below in the section, Is apo EmrE still able to interconvert?

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