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

Abstract

The 18-kilodalton translocator protein (TSPO), proposed to be a key player in cholesterol transport into mitochondria, is highly expressed in steroidogenic tissues, metastatic cancer, and inflammatory and neurological diseases such as Alzheimer's and Parkinson's. TSPO ligands, including benzodiazepine drugs, are implicated in regulating apoptosis and are extensively used in diagnostic imaging. We report crystal structures (at 1.8, 2.4, and 2.5 angstrom resolution) of TSPO from Rhodobacter sphaeroides and a mutant that mimics the human Ala(147)→Thr(147) polymorphism associated with psychiatric disorders and reduced pregnenolone production. Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand and conformational effects of the mutation. The three crystal structures show the same tightly interacting dimer and provide insights into the controversial physiological role of TSPO and how the mutation affects cholesterol binding.

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 A139Tmutant mimicking the human A147Tmutation. Dissociation constant (K_d) 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 WTcrystallized in a P2₁ space group. In all three crystal forms, the identical parallel dimer of RsTSPO was observed (Fig. 2). The A139Tmutant 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 WTand A139Tstructures.

Fig. 3. Structural comparison of WTand 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 WTand 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 F_o – F_c 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).