Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.

Autor: Spreacker PJ; Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, USA., Brousseau M; Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, USA., Hisao GS; Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, USA., Soltani M; Department of Chemistry, University of South Alabama, Mobile, Alabama, USA., Davis JH Jr; Department of Chemistry, University of South Alabama, Mobile, Alabama, USA., Henzler-Wildman KA; Department of Biochemistry, University of Wisconsin - Madison, Madison, Wisconsin, USA. Electronic address: henzlerwildm@wisc.edu.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2023 Feb; Vol. 299 (2), pp. 102805. Date of Electronic Publication: 2022 Dec 16.
DOI: 10.1016/j.jbc.2022.102805
Abstrakt: EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14 A and E14 B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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