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This article has been retracted
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Science 23 December 2005:
Vol. 310. no. 5756, pp. 1950 - 1953
DOI: 10.1126/science.1119776
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Reports
X-ray Structure of the EmrE Multidrug Transporter in Complex with a Substrate
Owen Pornillos,
Yen-Ju Chen,
Andy P. Chen and
Geoffrey Chang*
Department of Molecular Biology, The Scripps Institute, 10550 North Torrey Pines Road, CB-105, Jolla, CA 92037, USA.
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Fig. 1. Structure of the EmrE transporter in complex with TPP. (A) Stereoview cartoon representation of the asymmetric unit, composed of two EmrE subunits (subunit A in yellow and subunit B in green), and one bound TPP (red). Anomalous difference density shows the position of As (blue, contoured at 1 ), derived from crystals of EmrE with tetraphenylarsonium (an analog of TPP). Methionine positions are indicated by Se atoms (magenta, 4), derived from SeMet-EmrE-TPP crystals. Methionine side chains are shown explicitly and labeled; corresponding residues in subunit B and subunit A are distinguished by the asterisks. The coloring scheme in this panel is maintained throughout Figs. 1 2, 3. (B) Stereoview of the EmrE homodimer. The N and C termini of the two subunits are indicated. The boundaries of the lipid bilayer, deduced from the positions of aromatic groups, are shown by the gray lines. (C) Top view of the dimer, with the four transmembrane helices in each subunit labeled. The short helix connecting helices A2 and A3 is indicated by an asterisk. This view clearly shows that TPP is bound at the dimerization interface. The two Glu-14 residues are shown in red. (D) Best-fit superposition of subunits A (yellow) and B (green) (root mean square deviation of 3.5 Å over equivalent C positions). The first three helices form a left-handed bundle, whereas the fourth helices are positioned differently. (E) Crystal packing of EmrE-TPP. The lattice is stabilized by side-by-side transmembrane contacts and loop interactions, reminiscent of type I and 2D membrane protein crystals. A potential dimer of dimers is colored as above; the symmetry-related elements are colored gray. The unit cell is boxed in black. This view is perpendicular to the bc plane.
[View Larger Version of this Image (55K GIF file)]
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Fig. 2. A possible drug translocation pathway in the EmrE transporter. (A) Side view of the EmrE dimer, superimposed with a semitransparent surface rendering. The openings to the two pockets, facing opposite sides of the lipid bilayer, are indicated by arrows. Residues that have been shown by cysteine-scanning mutagenesis to be important for drug binding and transport (10, 11, 24–28) are indicated by spheres, using the following color scheme: blue, absolutely required for TPP binding; cyan, substitutions show partial TPP binding and impaired transport; magenta, residues that confer altered drug specificity when mutated. Residues in subunit B are indicated by asterisks. Note that EmrE mutagenesis alters two residues in the dimer, which are in nonequivalent positions. We have therefore only shown the positions that are close to the putative translocation pathway. Residues that are important for drug transport but have both copies removed from the pathway are shown in yellow (Leu-7, Tyr-60, Trp-63). These residues likely perform essential structural roles. (B) Close-up view of the bound drug, with protein, TPP, and As densities (contoured at 1). The three helices are B1 (left), A2 (middle), and A1 (right). The two Glu-14 residues are shown and colored red. The position of the phosphate atom is unambiguously defined by the anomalous As peak from the arsonium analog of TPP (blue), and the positions of the Glu-14 residues are relatively well defined based on the positions of Se atoms in Met-21, two helical turns away. Hydrophobic residues within van der Waals distance of TPP are also indicated. Close packing of TPP to helix A1 appears facilitated by the absence of a side chain in Gly-17. Mutation of this glycine to cysteine abolishes TPP binding (27).
[View Larger Version of this Image (52K GIF file)]
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Fig. 3. Comparison of the x-ray and EM structures of EmrE-TPP. (A) Independent superposition of EmrE subunits A and B in the x-ray structure with a cylinder model (colored gray) derived from the EM structure of EmrE-TPP (European Molecular Biology Laboratory–EBI accession code EMD-1087) (13). A unique match was found using two constraints: Three-helix bundles on opposite sides of the dimer were assumed to be helices 1 to 3, and the helix closest to the density attributed to TPP in the EM map was assumed to be helix 1. In this pseudo-atomic model, three helices have notably different tilt angles: A2, B2, and B3 (shown by red asterisks). The x-ray position of helices B2 and B3, which appear to move as a unit, is likely due to crystal packing interactions along the putative tetramerization interface (see also Fig. 1E). We speculate that the conformational change in helix A2 is relevant to the drug transport mechanism. (B and C) The TPP molecule is bound to different sites in the x-ray structure (C) and EM model (B), suggesting a possible mechanism for drug transport. The relative positions of the EmrE helices are indicated, viewing toward the binding pockets (in the same orientation as in Fig. 1C). The positions of the TPP molecules are shown by red circles.
[View Larger Version of this Image (18K GIF file)]
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Fig. 4. A potential mechanism for proton-dependent drug translocation by EmrE. For clarity, only the three putative gating helices (A1, A2, and B1) and two membrane-embedded Glu-14 side chains are shown explicitly. Drug substrates and protons are represented by the yellow sphere and red balls, respectively.
[View Larger Version of this Image (14K GIF file)]
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