Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism

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Science  30 Jan 2015:
Vol. 347, Issue 6221, pp. 555-558
DOI: 10.1126/science.1260590
  • Fig. 1 Structure and ligand binding affinity of RsTSPO WT and the A139T mutant.

    (A) Ligand binding affinities are shown for RsTSPO WT and the A139T mutant mimicking the human A147T mutation. Dissociation constant (Kd) values are obtained as described in (24): 10 ± 1 μM for PK11195 with WT, 42 ± 4 μM with A139T; 0.3 ± 0.01 μM for PpIX with WT, 1.9 ± 0.3 with A139T; and ~80 μM for cholesterol with WT, >300 μM with A139T. WT data are from (14), reproduced for comparison. (B) Overall structure of the A139T dimer. The position of the A139T mutation is labeled with a red asterisk and shown in sticks; the five transmembrane helices (TM-I to TM-V) are colored blue, green, wheat, orange, and red, respectively; and loop 1 (LP1) is colored teal. (C) Top view of (B). RsTSPO A139T crystallized in two different space groups (C2 and P212121) that have identical overall structures except for the flexible C terminus, whereas WT crystallized in a P21 space group. In all three crystal forms, the identical parallel dimer of RsTSPO was observed (Fig. 2). The A139T mutant in the C2 space group is shown here and used to discuss the major structural features of RsTSPO, as it has the highest resolution and most complete structure of RsTSPO. The N and C termini are labeled N/N′ and C/C′, respectively; the dashed lines highlight the approximate membrane region.

  • Fig. 2 Analysis of the dimer interface.

    (A) The dimer interface is primarily made up of TM-I and TM-III. Helices are colored the same as in Fig. 1 (blue, TM-I; gold, TM-III; orange, TM-IV) but are shown as space-filling models. TM-II and TM-IV make only minor contributions to the interface. Rotating the monomer by 180° about the dyad axis (blue dashed line) and overlaying it on top of itself creates the dimer. (B) Hydrophobic (white) and hydrogen bonding residues (blue, hydrogen bond donor atoms; red, hydrogen bond acceptor atoms) within the dimer interface. (C) Top view of a slab [between lines a and b in (A)]; residues forming strong interactions in the core of the dimer interface are labeled. The interface is essentially identical in all WT and A139T structures.

  • Fig. 3 Structural comparison of WT and A139T RsTSPO.

    Monomers of the WT (wheat) and A139T (green) are overlaid. (A) Overall structural alignment that highlights the degree and direction (arrows) of conformation change from WT to A139T for TM-II and TM-V. (B) Top view of the monomer, highlighting the side-chain rearrangements in sticks. Residue 139 is colored in blue; the CRAC site is colored in pink with two of three proposed critical residues (L142 and F144) highlighted in magenta. The prime symbol designates the mutant position. (C) Close-up side view of the potential ligand binding cavity, which reveals major differences in the conformations of LP1, TM-II, and TM-V between the WT and mutant proteins (fig. S7). The dotted yellow line denotes the location of unresolved LP1 in the WT structure (residues 29 to 40).

  • Fig. 4 Ligand binding and evidence for a transport pathway.

    Close-up view of the porphyrin binding site (A) with a porphyrin (pink) overlaid with feature enhanced map (FEM) omit map electron density (blue) and FoFc difference electron density (green), contoured at 1σ and ±3σ, respectively. A partially oxidized porphyrin is suggested by the spectrum (fig. S8A) that shows absence of a Soret band. (B) Surface groves on TSPO are occupied by monooleins and phospholipid (yellow). The CRAC site (red) is also interacting with lipids. Unusually long FEM omit map electron density (in blue and contoured at 1.0σ) extends from the porphyrin binding site to the bottom surface of the protein, beyond the bound phospholipid, suggesting an external transport pathway that involves both monomers of the dimer (see also fig. S9).

Supplementary Materials

  • Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism

    Fei Li, Jian Liu, Yi Zheng, R. Michael Garavito, Shelagh Ferguson-Miller

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S9
    • Table S1
    • Full Reference List

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