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

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...

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Autores principales: Iatmanen-Harbi, Soria, Senicourt, lucile, Papadopoulos, Vassilios, Lequin, Olivier, Lacapere, Jean-Jacques
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470738/
https://www.ncbi.nlm.nih.gov/pubmed/30901938
http://dx.doi.org/10.3390/ijms20061444
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author Iatmanen-Harbi, Soria
Senicourt, lucile
Papadopoulos, Vassilios
Lequin, Olivier
Lacapere, Jean-Jacques
author_facet Iatmanen-Harbi, Soria
Senicourt, lucile
Papadopoulos, Vassilios
Lequin, Olivier
Lacapere, Jean-Jacques
author_sort Iatmanen-Harbi, Soria
collection PubMed
description 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.
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spelling pubmed-64707382019-04-26 Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO Iatmanen-Harbi, Soria Senicourt, lucile Papadopoulos, Vassilios Lequin, Olivier Lacapere, Jean-Jacques Int J Mol Sci Article 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. MDPI 2019-03-21 /pmc/articles/PMC6470738/ /pubmed/30901938 http://dx.doi.org/10.3390/ijms20061444 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Iatmanen-Harbi, Soria
Senicourt, lucile
Papadopoulos, Vassilios
Lequin, Olivier
Lacapere, Jean-Jacques
Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title_full Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title_fullStr Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title_full_unstemmed Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title_short Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO
title_sort characterization of the high-affinity drug ligand binding site of mouse recombinant tspo
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470738/
https://www.ncbi.nlm.nih.gov/pubmed/30901938
http://dx.doi.org/10.3390/ijms20061444
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