Antibiotic resistance due to the activity of multidrug resistance (MDR) efflux pumps is one important mechanism of bacterial drug resistance. EmrE is one of the smallest known MDR transporters, and it has become a prototype for proton-coupled antiport. EmrE harnesses proton import to drive polyaromatic cation efflux in E. coli, thus conferring resistance to a broad range of drugs. We have previously performed the first quantitative measurement of conformational exchange between open-in and open-out states in a transporter by NMR. Our newest result, asymmetric protonation of EmrE, directly contradicts the long-accepted single-site alternating access model for coupled antiport of drugs and protons. It demonstrates how much is still unknown about this deceptively simply transporter and suggests that the well-known promiscuity of MDR efflux pumps may even be even greater. This project uses NMR spectroscopy and extensive liposomal transport assays to investigate the detailed molecular mechanism of EmrE activity. NMR offers a unique tool because protein conformational exchange and protonation events can be monitored separately and simultaneously, allowing novel insight into how proton and drug transport are coupled. Our goal is to combine quantitative biophysical data and functional assessment of the pH dependence and substrates properties that define EmrE activity to develop a novel model for proton-coupled antiport. We will also test different hypotheses for how proton- coupled antiport is achieved for such diverse substrates with different affinities and transport rates. The insights we gain will aid future efforts to combat bacterial antibiotic resistance due to MDR efflux.

Public Health Relevance

Active export of drug molecules by multidrug resistance (MDR) efflux proteins is one way that bacteria achieve antibiotic resistance. This project investigates how proton import is coupled to drug export in order to actively pump drugs out of the bacterial cytoplasm. The goal is to understand how multidrug resistance transporters are able to promiscuously export such diverse substrates so that future efforts will be able to more effectively target combat bacterial antibiotic resistance due to MDR efflux.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Garcia, Martha
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University of Wisconsin Madison
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United States
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Robinson, Anne E; Henderson, Jeffrey P; Henzler-Wildman, Katherine A (2018) A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates. Anal Biochem 549:130-135
Thomas, Nathan E; Wu, Chao; Morrison, Emma A et al. (2018) The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release. J Biol Chem 293:19137-19147
Robinson, Anne E; Thomas, Nathan E; Morrison, Emma A et al. (2017) New free-exchange model of EmrE transport. Proc Natl Acad Sci U S A 114:E10083-E10091
Morrison, Emma A; Robinson, Anne E; Liu, Yongjia et al. (2015) Asymmetric protonation of EmrE. J Gen Physiol 146:445-61
Morrison, Emma A; Henzler-Wildman, Katherine A (2014) Transported substrate determines exchange rate in the multidrug resistance transporter EmrE. J Biol Chem 289:6825-36
Dutta, Supratik; Morrison, Emma A; Henzler-Wildman, Katherine A (2014) EmrE dimerization depends on membrane environment. Biochim Biophys Acta 1838:1817-22
Dutta, Supratik; Morrison, Emma A; Henzler-Wildman, Katherine A (2014) Blocking dynamics of the SMR transporter EmrE impairs efflux activity. Biophys J 107:613-620
Morrison, Emma A; Henzler-Wildman, Katherine A (2012) Reconstitution of integral membrane proteins into isotropic bicelles with improved sample stability and expanded lipid composition profile. Biochim Biophys Acta 1818:814-20
Henzler-Wildman, Katherine (2012) Analyzing conformational changes in the transport cycle of EmrE. Curr Opin Struct Biol 22:38-43
Morrison, Emma A; DeKoster, Gregory T; Dutta, Supratik et al. (2012) Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481:45-50