Limits...
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

Structure of homodimeric TPP+-bound EmrE with pH-sensitive residues highlighted. Basic residues are colored blue, and acidic residues are colored red. TPP+ is not shown. Note that Glu14, shown as sticks with the protonatable oxygen represented as a ball, is the only charged residue in the TM regions. Yellow residues (Ala10, Gly17, and Ser43) were used for the pKa fits and are located near the Glu14 active site. Black residues are more remote from the active site but also fit to the same two pKa values determined using the yellow residues close to Glu14 (Protein Data Bank accession no. 3B5D).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Structure of homodimeric TPP+-bound EmrE with pH-sensitive residues highlighted. Basic residues are colored blue, and acidic residues are colored red. TPP+ is not shown. Note that Glu14, shown as sticks with the protonatable oxygen represented as a ball, is the only charged residue in the TM regions. Yellow residues (Ala10, Gly17, and Ser43) were used for the pKa fits and are located near the Glu14 active site. Black residues are more remote from the active site but also fit to the same two pKa values determined using the yellow residues close to Glu14 (Protein Data Bank accession no. 3B5D).

Mentions: Estimates of the pKa of Glu14 have been reported based on substrate binding and transport assays as a function of pH. The published Glu14 pKa values range from 6.8 to 8.5 (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b; Yerushalmi et al., 2001; Soskine et al., 2004; Adam et al., 2007), with more recent studies centering on a value of 7.3. Each of these experiments monitored a single bulk parameter reflecting EmrE function, either transport activity or substrate binding. These assays included both equilibrium and kinetic studies of transport or binding of radioactive substrates, proton release upon substrate binding monitored through direct pH measurement or pH-sensitive dyes, or substrate binding monitored through intrinsic tryptophan fluorescence. In all cases, the data were analyzed using a single pKa value, either as a simplifying assumption based on the stoichiometry of EmrE antiport (Rotem and Schuldiner, 2004; Soskine et al., 2004) and the “functional equivalence” of Glu14 in modification studies (Weinglass et al., 2005), or because the data could not justify the assignment of more than one pKa value (Adam et al., 2007). This long-held model of a single pKa implies that the two Glu14 residues, one from each subunit, are simultaneously protonated or deprotonated in a highly cooperative single event. However, consideration of the close proximity of the two ionizable Glu14 residues in the hydrophobic substrate-binding pocket of EmrE (Fig. 2) suggests the possibility of negative cooperativity in the deprotonation of the two Glu14 residues. Furthermore, non-identical pKa values for the two Glu14 residues might be expected as a result of the structural asymmetry of Glu14 (Chen et al., 2007; Lehner et al., 2008).


Asymmetric protonation of EmrE.

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

Structure of homodimeric TPP+-bound EmrE with pH-sensitive residues highlighted. Basic residues are colored blue, and acidic residues are colored red. TPP+ is not shown. Note that Glu14, shown as sticks with the protonatable oxygen represented as a ball, is the only charged residue in the TM regions. Yellow residues (Ala10, Gly17, and Ser43) were used for the pKa fits and are located near the Glu14 active site. Black residues are more remote from the active site but also fit to the same two pKa values determined using the yellow residues close to Glu14 (Protein Data Bank accession no. 3B5D).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Structure of homodimeric TPP+-bound EmrE with pH-sensitive residues highlighted. Basic residues are colored blue, and acidic residues are colored red. TPP+ is not shown. Note that Glu14, shown as sticks with the protonatable oxygen represented as a ball, is the only charged residue in the TM regions. Yellow residues (Ala10, Gly17, and Ser43) were used for the pKa fits and are located near the Glu14 active site. Black residues are more remote from the active site but also fit to the same two pKa values determined using the yellow residues close to Glu14 (Protein Data Bank accession no. 3B5D).
Mentions: Estimates of the pKa of Glu14 have been reported based on substrate binding and transport assays as a function of pH. The published Glu14 pKa values range from 6.8 to 8.5 (Muth and Schuldiner, 2000; Yerushalmi and Schuldiner, 2000b; Yerushalmi et al., 2001; Soskine et al., 2004; Adam et al., 2007), with more recent studies centering on a value of 7.3. Each of these experiments monitored a single bulk parameter reflecting EmrE function, either transport activity or substrate binding. These assays included both equilibrium and kinetic studies of transport or binding of radioactive substrates, proton release upon substrate binding monitored through direct pH measurement or pH-sensitive dyes, or substrate binding monitored through intrinsic tryptophan fluorescence. In all cases, the data were analyzed using a single pKa value, either as a simplifying assumption based on the stoichiometry of EmrE antiport (Rotem and Schuldiner, 2004; Soskine et al., 2004) and the “functional equivalence” of Glu14 in modification studies (Weinglass et al., 2005), or because the data could not justify the assignment of more than one pKa value (Adam et al., 2007). This long-held model of a single pKa implies that the two Glu14 residues, one from each subunit, are simultaneously protonated or deprotonated in a highly cooperative single event. However, consideration of the close proximity of the two ionizable Glu14 residues in the hydrophobic substrate-binding pocket of EmrE (Fig. 2) suggests the possibility of negative cooperativity in the deprotonation of the two Glu14 residues. Furthermore, non-identical pKa values for the two Glu14 residues might be expected as a result of the structural asymmetry of Glu14 (Chen et al., 2007; Lehner et al., 2008).

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