title: 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.
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, United States.
authors: Fei
authors: F
authors: Li
authors: Yan
authors: Y
authors: Xia
authors: Jens
authors: J
authors: Meiler
authors: Shelagh
authors: S
authors: Ferguson-Miller
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D020285:Cryoelectron Microscopy; D003460:Crystallization; D007546:Isoquinolines / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism* / Q000494:pharmacology; D051336:Mitochondrial Membranes / Q000378:metabolism; D055503:Protein Multimerization; D011524:Protoporphyrins / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D002352:Carrier Proteins; D007546:Isoquinolines; D008565:Membrane Proteins; D011524:Protoporphyrins; D011963:Receptors, GABA-A; D002784:Cholesterol; C028025:protoporphyrin IX; C037850:PK 11195
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