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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 mechanism of EmrE transport. EmrE is a homodimer composed of two subunits (blue and pink) oriented antiparallel to each other and in unique conformations (distinct shapes, labeled A and B). The subunits exchange conformations to switch between open-in and open-out forms. (A) In the standard single-site alternating access model, a single active site located between the two subunits alternates between binding protons or drug. Coupling between protons and drug is achieved by preventing exchange in the apo state and only allowing one substrate to bind at a time. (B) Drug-free EmrE has more states than represented in the basic model shown in A and most, if not all, of these states exchange.
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fig1: The mechanism of EmrE transport. EmrE is a homodimer composed of two subunits (blue and pink) oriented antiparallel to each other and in unique conformations (distinct shapes, labeled A and B). The subunits exchange conformations to switch between open-in and open-out forms. (A) In the standard single-site alternating access model, a single active site located between the two subunits alternates between binding protons or drug. Coupling between protons and drug is achieved by preventing exchange in the apo state and only allowing one substrate to bind at a time. (B) Drug-free EmrE has more states than represented in the basic model shown in A and most, if not all, of these states exchange.

Mentions: EmrE is a small multidrug resistance transporter in the inner membrane of Escherichia coli that exports polyaromatic cation substrates, conferring resistance to compounds of this chemical type. It is a secondary active transporter, importing protons to drive drug efflux. EmrE has long been thought to function by a single-site alternating access mechanism (Fig. 1 A; Yerushalmi and Schuldiner, 2000a). The highly conserved glutamate 14 (Glu14) is the only charged residue located in a TM helix. It is positioned in the middle of TM1 and is an essential residue necessary for binding both polyaromatic cation substrates and the counter-transported protons. As a result, Glu14 is central to coupling drug export to proton import to drive active efflux (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b; Soskine et al., 2004). It defines the “single-site” in the single-site alternating access model of EmrE transport activity. We have previously shown that drug-bound EmrE interconverts between inward- and outward-facing forms in a simple two-state exchange process with a rate that is determined by the identity of the bound substrate (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014). Here, we focus on the proton side of the transport cycle to investigate how EmrE performs coupled antiport of drug and protons (Fig. 1).


Asymmetric protonation of EmrE.

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

The mechanism of EmrE transport. EmrE is a homodimer composed of two subunits (blue and pink) oriented antiparallel to each other and in unique conformations (distinct shapes, labeled A and B). The subunits exchange conformations to switch between open-in and open-out forms. (A) In the standard single-site alternating access model, a single active site located between the two subunits alternates between binding protons or drug. Coupling between protons and drug is achieved by preventing exchange in the apo state and only allowing one substrate to bind at a time. (B) Drug-free EmrE has more states than represented in the basic model shown in A and most, if not all, of these states exchange.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: The mechanism of EmrE transport. EmrE is a homodimer composed of two subunits (blue and pink) oriented antiparallel to each other and in unique conformations (distinct shapes, labeled A and B). The subunits exchange conformations to switch between open-in and open-out forms. (A) In the standard single-site alternating access model, a single active site located between the two subunits alternates between binding protons or drug. Coupling between protons and drug is achieved by preventing exchange in the apo state and only allowing one substrate to bind at a time. (B) Drug-free EmrE has more states than represented in the basic model shown in A and most, if not all, of these states exchange.
Mentions: EmrE is a small multidrug resistance transporter in the inner membrane of Escherichia coli that exports polyaromatic cation substrates, conferring resistance to compounds of this chemical type. It is a secondary active transporter, importing protons to drive drug efflux. EmrE has long been thought to function by a single-site alternating access mechanism (Fig. 1 A; Yerushalmi and Schuldiner, 2000a). The highly conserved glutamate 14 (Glu14) is the only charged residue located in a TM helix. It is positioned in the middle of TM1 and is an essential residue necessary for binding both polyaromatic cation substrates and the counter-transported protons. As a result, Glu14 is central to coupling drug export to proton import to drive active efflux (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b; Soskine et al., 2004). It defines the “single-site” in the single-site alternating access model of EmrE transport activity. We have previously shown that drug-bound EmrE interconverts between inward- and outward-facing forms in a simple two-state exchange process with a rate that is determined by the identity of the bound substrate (Morrison et al., 2012; Morrison and Henzler-Wildman, 2014). Here, we focus on the proton side of the transport cycle to investigate how EmrE performs coupled antiport of drug and protons (Fig. 1).

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