Structural Integrity of the A147T Polymorph of Mammalian TSPO

Abstract

Ligands of the transmembrane protein TSPO are used for imaging of brain inflammation, but a common polymorphism in TSPO complicates their application to humans. Here we determined the three-dimensional structure and side-chain dynamics of the A147T polymorph of mammalian TSPO in complex with the first-generation ligand PK11195. We show that A147T TSPO is able to retain the same structural and dynamic profile as the wild-type protein and thus binds PK11195 with comparable affinity. Our study is important for the design of more potent diagnostic and therapeutic ligands of TSPO.

Keywords: NMR spectroscopy; dynamics; membrane proteins; polymorphism; protein structures.

Figure 1

3D structure of A147T-mTSPO in complex with (R)-PK11195. (A) ¹H-¹⁵N HSQC spectra of A147T-mTSPO (orange) and wild-type mTSPO (silver) in complex with PK11195. Selected resonances are labelled. (B) NMR ensemble of the 20 lowest-energy structures of A147T-mTSPO (PDB id: 2N02). PK11195 is depicted by sticks. The cytosolic helix is labelled as α’. (C) Comparison of the 3D structures of A147T (orange) and wild-type mTSPO (silver; PDB id: 2MGY) in complex with PK11195. (D) Detailed view of the local structure around position 147 in A147T-mTSPO (orange) and mTSPO (silver) both bound to PK11195.

Figure 2

Chemical shift differences between A147T-mTSPO and wild-type mTSPO, both in complex with (R)-PK11195. (A,B) ¹H/¹⁵N chemical shift perturbation (CSP) as a function of residue number (A) and mapped onto the 3D structure of A147T-mTSPO in complex with (R)-PK11195 (B). In (A), the location of helices is shown by black bars and the site of mutation is labelled. In (B), threonine at position 147 is colored black. (C,D) CSP of tryptophan imidazole protons upon substitution of alanine by threonine at position 147 as a function of residue number (C) and mapped onto the 3D structure of A147T-mTSPO in complex with (R)-PK11195 (D).

Figure 3

Dynamics of methyl groups of A147T-mTSPO in complex with PK11195. (A) Cross-correlated relaxation rates (σ_obs) between dipolar couplings in methyl groups of A147T-mTSPO (orange) and wild-type mTSPO (silver). (B) Mapping of σ_obs values from (a; orange) onto the 3D structure of the A147T-mTSPO-PK11195 complex.

Figure 4

Structural integrity of the cholesterol recognition sequence in mammalian TSPO. (A) Detailed view of the conformation of the cholesterol recognition sequence in A147T-mTSPO (orange) and wild-type mTSPO (silver; PDB id: 2MGY) both in complex with PK11195. Side chains of selected resonances are shown. (B) Superposition of ¹H-¹⁵N HSQC spectra of A147T-mTSPO (orange) and wild-type mTSPO (silver) in the absence of PK11195. (C) Comparison of the 3D structures of the A147T-mTSPO-PK11195 (orange) and mTSPO-PK11195 (silver: PDB id: 2MGY) complexes with those of the tryptophan-rich sensory proteins from Bacillus cereus (green: PDB id: 4RYQ) and from Rhodobacter sphaeroides (magenta; PDB id: 4UC1:A) and its A139T variant (light blue: PDB id: 4UC3:A). In wild-type RsTspO the TM1-TM2 loop was not observed and is therefore approximated by dots. The orientation of the side chains of Y152 and R156 in A147T-mTSPO, which are essential for cholesterol binding, is similar to the side chain orientation of the homologous residues in wild-type BcTspO and A139T RsTspO, but not in wild-type RsTspO.

Figure 5

Alignment of mammalian TSPO sequences. A147 is marked in yellow. Residues in helical regions are colored orange.