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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

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).
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fig11: 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).

Mentions: The single-site alternating access model predicts that truly apo EmrE should not interconvert between open-in and open-out. In theory, EmrE at high pH in the absence of drug should exist in a true apo state. However, based on the pKa values determined above, we estimate that at pH 8.8 (the highest pH studied), EmrE is ∼80% fully deprotonated and 20% singly protonated, with <1% doubly protonated. At this pH, two peaks are observed that correspond to subunits A and B in the asymmetric EmrE homodimer. Unexpectedly, additional cross peaks are clearly visible at this pH in the spectrum at 45°C (Figs. 3 A and 5 A). These cross peaks (Fig. 5 A, peaks marked with “x”) are visible at pH 8.0 and above and are aligned with the proton and nitrogen chemical shifts of the major peaks to form a square pattern (Fig. 11 A). This indicates conformational exchange occurring on a tens per second timescale. We have previously observed similar peak patterns in TROSY-HSQC spectra of EmrE bound to ethyltriphenylphosphonium+ (Morrison and Henzler-Wildman, 2014) as a result of interconversion between open-in and open-out forms at a rate of 25 s−1. We demonstrated that cross peaks appear in the TROSY-HSQC spectrum when rates are fast enough for significant exchange to occur during the 11-ms back-transfer portion of the NMR pulse sequence (Morrison and Henzler-Wildman, 2014). Using this 11-ms effective mixing time, we can estimate a conformational exchange rate of 50 s−1 for drug-free EmrE at pH 8.8, 45°C, using the intensities of the well-resolved Ala10 and Gly9 auto and cross peaks. Using the pKa values determined above, at pH 8.8 EmrE is ∼80% fully deprotonated and 20% monoprotonated, and there is essentially no doubly protonated EmrE. The exchange rate is faster, 75 s−1, at pH 8.4 when EmrE is 60–65% deprotonated. Collectively, the data are consistent with the idea that EmrE can exchange in more than just the doubly protonated state.


Asymmetric protonation of EmrE.

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

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

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

fig11: 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).
Mentions: The single-site alternating access model predicts that truly apo EmrE should not interconvert between open-in and open-out. In theory, EmrE at high pH in the absence of drug should exist in a true apo state. However, based on the pKa values determined above, we estimate that at pH 8.8 (the highest pH studied), EmrE is ∼80% fully deprotonated and 20% singly protonated, with <1% doubly protonated. At this pH, two peaks are observed that correspond to subunits A and B in the asymmetric EmrE homodimer. Unexpectedly, additional cross peaks are clearly visible at this pH in the spectrum at 45°C (Figs. 3 A and 5 A). These cross peaks (Fig. 5 A, peaks marked with “x”) are visible at pH 8.0 and above and are aligned with the proton and nitrogen chemical shifts of the major peaks to form a square pattern (Fig. 11 A). This indicates conformational exchange occurring on a tens per second timescale. We have previously observed similar peak patterns in TROSY-HSQC spectra of EmrE bound to ethyltriphenylphosphonium+ (Morrison and Henzler-Wildman, 2014) as a result of interconversion between open-in and open-out forms at a rate of 25 s−1. We demonstrated that cross peaks appear in the TROSY-HSQC spectrum when rates are fast enough for significant exchange to occur during the 11-ms back-transfer portion of the NMR pulse sequence (Morrison and Henzler-Wildman, 2014). Using this 11-ms effective mixing time, we can estimate a conformational exchange rate of 50 s−1 for drug-free EmrE at pH 8.8, 45°C, using the intensities of the well-resolved Ala10 and Gly9 auto and cross peaks. Using the pKa values determined above, at pH 8.8 EmrE is ∼80% fully deprotonated and 20% monoprotonated, and there is essentially no doubly protonated EmrE. The exchange rate is faster, 75 s−1, at pH 8.4 when EmrE is 60–65% deprotonated. Collectively, the data are consistent with the idea that EmrE can exchange in more than just the doubly protonated state.

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