Structure, dynamics, and substrate-induced conformational changes of the multidrug transporter EmrE in liposomes

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.

FIGURE 1.

Growth phenotypes of cells expressing EmrE single cysteine mutants. Saturated cultures of each mutant were diluted by a factor of 10⁶, 10³, and 10 and spotted on agar plates containing ethidium bromide as described under “Experimental Procedures.” The height of the bar indicates the maximal dilution at which cell growth was observed. * indicates mutants that did not support a resistance phenotype.

FIGURE 2.

Changes in TPP⁺ binding affinity of selected spin-labeled EmrE detected by the level of Trp quenching. For a number of residues (e.g. 44), binding required high concentration of EmrE and TPP⁺ indicating reduction in affinity due to the cysteine substitution and/or attachment of the spin label.

FIGURE 3.

Analysis of EmrE dimeric assembly by size exclusion chromatography. A, spin labeling of selected residues in TM4 leads to changes in the retention time indicative of aggregation. B, detergent nonyl glucoside (NG) destabilizes the apo EmrE resulting in dissociation to a monomer. The inset is a cut out from an SDS-PAGE confirming the protein identity in each SEC peak. βddm, β-dodecyl maltoside.

FIGURE 4.

Accessibility profiles of apo EmrE. Red, Π(O₂); blue, Π(NiEDDA). Both parameters show periodic variation as a function of residue number in the regions of the TM helices. The dashed lines indicate helix and loop boundaries on the basis of the crystal structure assignment.

FIGURE 5.

Sequence-specific environmental parameters in the apo (color) and TPP⁺ bound intermediates (gray). A, mobility parameter (ΔH₀)⁻¹. B, accessibility to molecular O₂, and C, accessibility to NiEDDA. The regions of accessibility changes are highlighted in yellow.

FIGURE 6.

EPR line shape changes upon TPP⁺ binding reveal changes in packing and proximity along the interfaces of TM1–3. For A–C, a close up view of the structure highlighting the spin-labeled residues is shown. A and B, the bound TPP molecule is also visible. All spectra were normalized to the same number of spins and then scaled to reveal the details of the line shapes.

FIGURE 7.

Π(O₂) (A) and Π(NiEDDA) (B) were mapped onto a ribbon representation of the two EmrE monomers. The second monomer is shown in a surface rendering. Selected residues from each TM are shown to provide markers and highlight the structural asymmetry between the two monomers.

FIGURE 8.

Model of TM3 based on the EPR data (yellow) superimposed on a ribbon representation of the helix in the crystal structure. It assumes a 2-fold symmetry axis near the middle of the bilayer. This configuration satisfies the constraints of proximity between residues 60 and 64. The arrows indicate movement of the C-terminal tail that allows residues at the C terminus and in the loop linking TM3 to -4 to sample the membrane and aqueous environments.