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Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

ABSTRACT: Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecular mechanism of their effects remain controversial, in part because of the lack of an atomic-resolution structure. We expressed and purified the homologue of mammalian TSPO from Rhodobacter sphaeroides (RsTSPO), as well as a mutant form in a proposed drug binding loop, RsTSPOW38C. We characterized their binding behaviors with endogenous ligands and a series of compounds that affect apoptosis by using a sensitive tryptophan fluorescence quenching assay. Our results show that RsTSPO behaves as a dimer in the purified state and binds with low micromolar affinity to many of these ligands, including retinoic acid, curcumin, and a known Bcl-2 inhibitor, gossypol, suggesting a possible direct role for TSPO in their regulation of apoptosis. A computational model of the RsTSPO dimer is constructed using EM-Fold, Rosetta, and a cryo-electron microscopy density map. Binding behaviors of known ligands are discussed in the context of the model with respect to regions that may be involved in binding.

SUBMITTER: Li F

PROVIDER: S-EPMC3756528 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

Li Fei F Xia Yan Y Meiler Jens J Ferguson-Miller Shelagh S

Biochemistry 20130816 34

Translocator Protein 18 kDa (TSPO), previously known as the peripheral-type benzodiazepine receptor (PBR), is a mitochondrial outer membrane protein that has been identified as a key player in cholesterol and porphyrin transport, apoptotic signaling, and cancer development, as well as neurological inflammation and disease. Despite a number of TSPO ligands whose effects have been studied with respect to these varied biological activities, the nature of their interactions with TSPO and the molecul ...[more]

PMID: 23952237

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Project description:UNLABELLED:11C-PBR28 PET can detect the 18-kDa translocator protein (TSPO) expressed within macrophages. However, quantitative evaluation of the signal in brain tissue from donors with multiple sclerosis (MS) shows that PBR28 binds the TSPO with high affinity (binding affinity [Ki], ?4 nM), low affinity (Ki, ?200 nM), or mixed affinity (2 sites with Ki, ?4 nM and ?300 nM). Our study tested whether similar binding behavior could be detected in brain tissue from donors with no history of neurologic disease, with TSPO-binding PET ligands other than 11C-PBR28, for TSPO present in peripheral blood, and with human brain PET data acquired in vivo with 11C-PBR28. METHODS:The affinity of TSPO ligands was measured in the human brain postmortem from donors with a history of MS (n=13), donors without any history of neurologic disease (n=20), and in platelets from healthy volunteers (n=13). Binding potential estimates from thirty-five 11C-PBR28 PET scans from an independent sample of healthy volunteers were analyzed using a gaussian mixture model. RESULTS:Three binding affinity patterns were found in brains from subjects without neurologic disease in similar proportions to those reported previously from studies of MS brains. TSPO ligands showed substantial differences in affinity between subjects classified as high-affinity binders (HABs) and low-affinity binders (LABs). Differences in affinity between HABs and LABs are approximately 50-fold with PBR28, approximately 17-fold with PBR06, and approximately 4-fold with DAA1106, DPA713, and PBR111. Where differences in affinity between HABs and LABs were low (?4-fold), distinct affinities were not resolvable in binding curves for mixed-affinity binders (MABs), which appeared to express 1 class of sites with an affinity approximately equal to the mean of those for HABs and LABs. Mixed-affinity binding was detected in platelets from an independent sample (HAB, 69%; MAB, 31%), although LABs were not detected. Analysis of 11C-PBR28 PET data was not inconsistent with the existence of distinct subpopulations of HABs, MABs, and LABs. CONCLUSION:With the exception of 11C-PK11195, all TSPO PET ligands in current clinical application recognize HABs, LABs, and MABs in brain tissue in vitro. Knowledge of subjects' binding patterns will be required to accurately quantify TSPO expression in vivo using PET.

Dataset Information

Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

Publications

Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides.

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