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Edited by Michael F. Summers, Howard Hughes Medical Institute, University of Maryland, Baltimore, MD, and approved October 15, 2017 (received for review May 25, 2017)
Significance
EmrE facilitates Escherichia coli multidrug resistance by coupling drug efflux to proton import. This antiport mechanism has been thought to occur via a pure-exchange model, which achieves coupled antiport by restricting when the single binding pocket can alternate access between opposite sides of the membrane. We test this model using NMR titrations and transport assays and find it cannot account for EmrE antiport activity. We propose a new free-exchange model of antiport with fewer restrictions that better accounts for the highly promiscuous nature of EmrE drug efflux. This model expands our understanding of proton-coupled transport and has implications for both transporter design and drug development.
Abstract
EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.
Footnotes
↵1Present address: Division of Infectious Diseases, Department of Internal Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO 63110.
↵2Present address: Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242.
↵3Present address: Biotherapeutics Pharmaceutical Sciences Research and Development, Pfizer Inc., Chesterfield, MO 63017.
- ↵4To whom correspondence should be addressed. Email: henzlerwildm{at}wisc.edu.
Author contributions: A.E.R., N.E.T., E.A.M., and K.A.H.-W. designed research; A.E.R., N.E.T., E.A.M., B.M.B., and K.A.H.-W. performed research; A.E.R., N.E.T., E.A.M., B.M.B., and K.A.H.-W. analyzed data; and A.E.R., N.E.T., E.A.M., and K.A.H.-W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1708671114/-/DCSupplemental.
Published under the PNAS license.
Free-exchange model of EmrE transport
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- Biological Sciences
- Biophysics and Computational Biology
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