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Antiparallel EmrE exports drugs by exchanging between asymmetric structures

Abstract

Small multidrug resistance transporters provide an ideal system to study the minimal requirements for active transport. EmrE is one such transporter in Escherichia coli. It exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy has surrounded the topology of the EmrE homodimer. Here we show that asymmetric antiparallel EmrE exchanges between inward- and outward-facing states that are identical except that they have opposite orientation in the membrane. We quantitatively measure the global conformational exchange between these two states for substrate-bound EmrE in bicelles using solution NMR dynamics experiments. Förster resonance energy transfer reveals that the monomers within each dimer are antiparallel, and paramagnetic relaxation enhancement NMR experiments demonstrate differential water accessibility of the two monomers within each dimer. Our experiments reveal a ‘dynamic symmetry’ that reconciles the asymmetric EmrE structure with the functional symmetry of residues in the active site.

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Figure 1: Conformational interconversion and symmetry in the single-site alternating access model of EmrE transport.
Figure 2: TPP + -bound EmrE interconverts between two conformations.
Figure 3: EmrE is an antiparallel homodimer.
Figure 4: EmrE has asymmetric water accessibility.

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Acknowledgements

We thank Y. Liu for assistance replicating the ITC data. We thank J. Villali for assistance growing isotopically labelled EmrE. We are grateful to Y. Li and A. Palmer for providing pulse programs and G. Chang for providing the EmrE expression plasmid. This work was supported by the National Institutes of Health (1R01GM095839) and the Searle Scholars Program (K.H.W.), the US Department of Energy, Office of Basic Energy Sciences (D.K.), the Howard Hughes Medical Institute (D.K. and T.H.), and an NSF graduate research fellowship to E.M. (DGE-1143954).

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Contributions

E.M. and K.H.W. optimized EmrE sample preparation, performed ITC experiments and recorded two-dimensional NMR spectra under different conditions. E.M. and G.D. performed the bulk FRET experiments. S.D. and R.V. performed the single molecule FRET experiments with guidance from T.H. G.D. and M.C. optimized the modified ZZ-exchange NMR experiment. M.C. collected the ZZ-exchange data and K.H.W. analysed it. G.D. performed the paramagnetic relaxation enhancement NMR experiments. A.B., E.M., G.D. and K.H.W. contributed to assignments. D.K. mentored initial project development. K.H.W. conceived the project and wrote the manuscript.

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Correspondence to Katherine A. Henzler-Wildman.

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The file contains Supplementary Figures 1-15 with legends, full-page versions of Figures 1c, 2a and 4a from the main paper, Supplementary Table 1, Supplementary Discussion and additional references. (PDF 7376 kb)

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Morrison, E., DeKoster, G., Dutta, S. et al. Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481, 45–50 (2012). https://doi.org/10.1038/nature10703

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