Abstract
EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.
Highlights
Grant GM077659 from the NIGMS. □S The on-line version of this article contains supplemental Figs. 1– 6. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: 2215 Garland Ave., 741
Much of the current mechanistic understanding of small multidrug resistance transporters has emerged from seminal studies in the laboratory of Schuldiner and co-workers (4 –7)defining fundamental steps in the transport cycle of Escherichia coli EmrE
Biochemical Analysis of EmrE Mutants—To assess the transport activity of the EmrE cysteine mutants, we determined whether they conferred drug resistance on transformed E. coli cells
Summary
Grant GM077659 from the NIGMS. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1– 6. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: 2215 Garland Ave., 741. With four predicted transmembrane ␣-helices and no significant extramembrane domain [2, 3] They function as dimers coupling translocation of positively charged hydrophobic substrates out of the cell to the inward movement of protons. EmrE structures, determined from twoand three-dimensional crystals [15,16,17], provide compelling evidence supporting antiparallel orientation of the monomers. A body of biochemical data supports a parallel orientation of the dimer. These include the design and construction of functional EmrE chimeras where monomers are linked by short polar loops not favored energetically to cross the bilayer [20]. Limited structural constraints derived from EPR analysis of spin-labeled EmrE in TM3 were interpreted as consistent with a parallel orientation [19]
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