Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO.

Soria Iatmanen-Harbi,Jean-Jacques Lacapere,Lucile Senicourt,Vassilios Papadopoulos,Olivier Lequin

doi:10.3390/ijms20061444

Abstract

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

Highlights

The 18 kDa translocator protein (TSPO), previously named PBR for peripheral-type benzodiazepine receptor [1], is an evolutionarily conserved membrane protein [2] located in eukaryotic cell mitochondria
They involve 10 amino acids (A23, V26, L49, A50, I52, W107, A110, L114, A147 and L150, displayed in orange in Figure 3, except for W107 shown in green), but an atomic structure analysis suggested the involvement of 5 additional amino acids (R46, W53, W95, D111 and W143), which are at a short distance from the bound PK 11195
Y34 present in TSPO1 that we describe as important for targeting PK 11195 to the binding pocket is absent in TSPO2

Summary

Introduction

The 18 kDa translocator protein (TSPO), previously named PBR for peripheral-type benzodiazepine receptor [1], is an evolutionarily conserved membrane protein [2] located in eukaryotic cell mitochondria. Many TSPO ligands belonging to different chemical classes have been identified over the last decades [4], but overly complex binding profiles likely due to the number of genetic variants [7] as well as the lack of atomic structures have not permitted the optimization of drug design [8]. The atomic structure of recombinant mouse TSPO (rec-mTSPO) was determined by NMR [9] (PDB ID-2MGY), after stabilization by its high-affinity drug ligand, PK 11195 [10]. Various mutations and deletions have been reported in mammalian and bacterial species [11,12,13,14], suggesting the involvement of the five transmembrane helices and the cytosolic loops, which were confirmed by the atomic structures determined for mammalian and bacterial TSPO [9,15,16]. The most significant amino acid differences are observed when comparing TSPO (TSPO1) to its paralogous gene product TSPO2 that correlates with the observed differences in the PK 11195 binding affinities for these proteins [17]

Methods

Findings

Discussion

Conclusion