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. 2019 Dec 5;8:e51913. doi: 10.7554/eLife.51913

Figure 3. Structure of inhibitor complexes.

(A-B) Close-up views of the crystal structure of EcoDMT in complex with Br-BIT determined at 3.8 Å resolution viewed from within the membrane. The refined 2Fo-Fc electron density is shown as blue mesh. The position of Br-BIT is defined by the anomalous difference density of the Br-atom (shown as magenta mesh, contoured at 7σ) and by residual density in the 2Fo-Fc omit map (dark yellow mesh). C-D, Ribbon representations of the EcoDMT structure viewed from within the membrane (C) and from the extracellular side (D). The Mn2+ binding site is indicated with a red asterisk. The molecular surfaces are represented as gray meshes. The C-terminal sub-domain (α-helices 6–12) is shown in dark blue, α-helices 11 and 12 in magenta. (E) Position of Br-BIT in the binding pocket (gray mesh) of EcoDMT. (F) Detailed view of the residues in contact distance to Br-BIT. (G) Position of Br-BIT in the binding pocket (gray mesh) of a homology model of human DMT1. (H) Potential interactions of Br-BIT with the homology model of human DMT1. A-H, The proximal (p) isothiourea group is close to the metal ion coordinating residues (marked with a black asterisk) and the distal (d) isothiourea group is in proximity to α-helix 11.

Figure 3.

Figure 3—figure supplement 1. Inhibitor binding to EcoDMT.

Figure 3—figure supplement 1.

(A) Anomalous difference density of five datasets of EcoDMT/Br-BIT complexes. The numbering of datasets is as in Table 3. The inhibitor is shown as stick, the anomalous difference density of the Br-atom (1, 3–5 contoured at 6σ, 2 at 4σ) as magenta mesh. (B) Schematic depiction of interactions between Br-BIT and EcoDMT (left) as observed in the complex structure compared to the assumed interactions between the metal ion (brown sphere) and EcoDMT (right). Hydrogen bonds and ionic interactions are indicated by dashed lines and hydrophobic contacts are represented by an arc. The 2D plots were generated using LIGPLOT (Wallace et al., 1995). (C) Electrostatic potential within the inhibitor binding site of EcoDMT as obtained from a numerical solution of the Poisson-Boltzmann equation calculated from the protein in the absence of the inhibitor. Red bars show the potential measured at the respective center of either the proximal or distal isothiourea group obtained from the coordinates of the refined inhibitor complex. The negative potential underlines the attractive electrostatic environment for interactions with the positively charged groups.
Figure 3—figure supplement 2. Homology model of hDMT1.

Figure 3—figure supplement 2.

(A) Sequence alignment of hDMT1 (UniProtID: P49281-3) and EcoDMT (UniProtID: E4KPW4). Secondary structure elements of EcoDMT are indicated below. Identical residues are highlighted in green, similar residues in yellow. Red spheres mark residues of the metal ion binding site, green spheres residues mutated in hDMT1 and EcoDMT and blue spheres residues mutated in hDMT1. (B) Stereo view of a superposition of EcoDMT with a homology model of hDMT1. The location of the metal ion binding site is indicated by a black sphere. (C) Electrostatic potential within the inhibitor binding site of hDMT1 as obtained from a numerical solution of the Poisson-Boltzmann equation calculated from a homology model of the protein in the absence of the inhibitor. Green bars show the potential measured at the respective center of either the proximal or distal isothiourea group obtained from the coordinates of a modeled inhibitor complex. The negative potential underlines the attractive electrostatic environment for interactions with the positively charged groups.