Active export of drug molecules by multidrug resistance (MDR) efflux proteins is one important mechanism of bacterial drug resistance. EmrE is one of the smallest known MDR transporters, making it an ideal system to study the minimum requirements for MDR efflux. EmrE couples proton import to polyaromatic cation export in E. coli, thus conferring resistance to a broad range of drugs of this type. Although the details are not known, protein conformational change must occur during transport, allowing alternating access to either side of the membrane in response to substrate binding. This project investigates the transport mechanism of EmrE using solution NMR spectroscopy to determine the structures of the multiple states in the transport cycle along with the kinetics of conformational exchange between those states. NMR offers a unique tool to obtain this information, since kinetic and structural data are measured simultaneously at multiple sites across the protein with atomic resolution. Quantitative measurement of the dynamics of EmrE solubilized in fast-tumbling bicelles will be performed using modern solution NMR and single molecule FRET. Single molecule FRET provides a complementary method that can detect details obscured by population averaging and can be used both in bicelles and in liposomes. This data will be compared with standard binding and transport assays to experimentally test two important hypotheses in the single-site alternating access model of coupled antiport with relevance to multidrug efflux: (i) conformational inter- conversion between inward- and outward-facing states is the rate-limiting step for transport, and (ii) binding substrates with different affinities leads to a different energy landscape of the bound state, and thus different rates of conformational interconversion. This knowledge will improve our understanding of secondary active transport and aid 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 bacteria achieve antibiotic resistance. This project investigates the transport mechanism of the small multidrug resistance transporter, EmrE, using solution NMR spectroscopy and single molecule FRET. It will improve our understanding of multidrug recognition and secondary active transport mechanisms, aiding future efforts to combat bacterial antibiotic resistance due to MDR efflux.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM095839-04
Application #
8681469
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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 107:613-20
Morrison, Emma A; DeKoster, Gregory T; Dutta, Supratik et al. (2012) Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481:45-50
Henzler-Wildman, Katherine (2012) Analyzing conformational changes in the transport cycle of EmrE. Curr Opin Struct Biol 22:38-43
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