Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

Abstract

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO. An ortholog in the photosynthetic bacterium Rhodobacter was independently discovered as the tryptophan-rich sensory protein (TspO) and found to play a role in the response to changes in oxygen and light conditions that regulate photosynthesis and respiration. As part of this highly conserved protein family found in all three kingdoms, the rat TSPO is able to rescue the knockout phenotype in Rhodobacter, indicating functional as well as structural conservation. Recently, a major breakthrough in the field was achieved: the determination of atomic-resolution structures of TSPO from different species by several independent groups. This now allows us to reexamine the function of TSPO with a molecular perspective. In this review, we focus on recently determined structures of TSPO and their implications for potential functions of this ubiquitous multifaceted protein. We suggest that TSPO is an ancient bacterial receptor/stress sensor that has developed additional interactions, partners, and roles in its mitochondrial outer membrane environment in eukaryotes.

Figure 1

Comparison of TSPO monomers from different species. The high-resolution crystal structure of RsTSPO A139T (PDB entry 4UC1) is colored in discrete rainbow and shown as a cartoon in panel A: TM1, blue; LP1, teal; TM2, green; TM3, wheat; TM4, orange; TM5, red. (B) Comparison of the structures of RsTSPO and BcTSPO (PDB entry 4RYQ). RsTSPO is colored the same as in panel A and shown partially transparent, while the crystal structure of BcTSPO is colored magenta and shown as a tube. (C) Comparison of the structures of RsTSPO and mTSPO (PDB entry 2MGY). RsTSPO is colored the same as in panel A and shown partially transparent, while the NMR structure of mTSPO is colored black and shown as a tube.

Figure 2

External loop, LP1, which has a defined, conserved structure across evolution and interacts with TM5 and TM2. Residues on LP1 and interacting pairs predicted by covariance analysis are shown as sticks and colored in matching colors, with red having the highest-confidence pairing across evolution, followed by magenta and orange. K36, D32, and R43 form predicted salt bridges (yellow dotted lines) with backbone and side chains maintaining a defined structure for LP1. W39 and G141 also closely interact as predicted. Conservation of interacting pairs suggests the structure of LP1 and its interaction with TM5 and TM2 is an important structural element for TSPO across different species and may play an essential functional role (figure created in Pymol from PDB entry 4UC1).

Figure 3

RsTSPO and BcTSPO form different dimers. A sequence alignment of TSPOs from human, mouse, rat, R. sphaeroides, and B. cereus is shown in panel A, while the two different dimer assemblies for RsTSPO (B and C; PDB entry 4UC1) and BcTSPO (D and E; PDB entry 4RYJ) are shown in side views and top views. In panel A, residues on the dimer interface of RsTSPO are labeled as cyan triangles while residues for the BcTSPO interface are labeled as cyan dots.

Figure 4

Ligand binding sites for TSPO from different species. Ligand binding sites in currently available structures of different species are positioned in the central cavity but have different interacting residues. TSPO proteins are colored wheat, while ligands (PK11195 and porphyrin) are colored green. Side chains interacting with the ligands are shown as orange sticks. (A) BcTSPO with PK11195 bound (PDB entry 4RYI). (B) mTSPO with PK11195 bound (PDB entry 2MGY). (C) RsTSPO with a representative porphyrin bound (PDB entry 4UC1). (D) As in panel C but from the top to show the position of residue P47, which is a histidine and binds heme in plant TSPO.

Figure 5

Favored cholesterol binding site predicted in TSPO. (A) A CholMine-predicted cholesterol binding position (black sticks) is shown on the crystal structure of RsTSPO, where the A chain of RsTSPO A139T (PDB entry 4UC1) is represented with main chain ribbon and side chain sticks for the LAF and CRAC motif residues (A136, T137, and A138 colored yellow and L142, F144, and R148 colored orange). The main chain of TSPO is colored according to crystal temperature factor values, with blue indicating low-mobility, green moderate-mobility, and red high-mobility regions. (B) The AB dimer of RsTSPO A139T is shown in surface representation. The most favorable position for cholesterol binding is shown in black space-filling representation, located in the vicinity of the CRAC site (yellow) and LAF site (orange). Monoolein lipids are colored cyan; a phospholipid is colored blue, and a porphyrin-type ligand is colored red. In panel A, one partial monoolein that sits parallel with the predicted cholesterol binding site is shown as sticks, while in panel B, all crystallographically observed lipids are shown in space-filling representation.

Figure 6

Observed lipid binding sites. Lipids are consistently observed in similar positions in different crystal structures of RsTSPO grown under different conditions (shown as sticks). Lipids observed in the 1.8 Å structure (PDB entry 4UC1) are colored blue (AB dimer) and cyan (CC′ dimer), while lipids observed in the 2.4 Å structure (PDB entry 5DUO) are colored orange (AB dimer) and yellow (CC′ dimer).

Figure 7

TSPO structure that resembles GPCR structure with a similar “toggle switch”. TSPO resembles GPCRs in the overall architecture with the extracellular loop (teal) on top of the central ligand binding site. A highly conserved WxPxF motif was also found within a transmembrane helix in RsTSPO (B), similar to that identified in the structure of β₂-adrenergic receptor (A). ProFlex analysis of RsTSPO A139T indicates that the WxPxF motif in RsTSPO creates a flexible hinge, centered on the tryptophan, between two segments of TM3 (C). WxPxF motifs are colored magenta in panels A and B, while panel C is colored by main chain flexibility based on ProFlex analysis.^, ProFlex analysis was performed on a ligand-free version of the A139T crystal structure (chain B of PDB entry 4UC1). It allows identification of regions of the TSPO structure with different degrees of stability: blue regions are highly constrained and mutually rigid, due to the presence of a dense network of hydrophobic interactions and hydrogen bonds; gray regions have borderline stability due to a weak network of hydrophobic and hydrogen bond interactions; orange regions are more flexible; and red regions are highly flexible, with few stabilizing noncovalent interactions. Note that the relative flexibility of the helix 1–2 and 3–4 loops and the free C-terminal region of RsTSPO A139T (loops and short helix at the top of the figure) predicted by ProFlex are very similar to the flexibility of these regions indicated by crystallographic temperature factors in Figure 5A.