Evidence for crosstalk between the aryl hydrocarbon receptor and the translocator protein in mouse lung epithelial cells.

Cellular homeostasis requires the use of multiple environmental sensors that can respond to a variety of endogenous and exogenous compounds. The aryl hydrocarbon receptor (AHR) is classically known as a transcription factor that induces genes that encode drug metabolizing enzymes when bound to toxicants such as 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD). The receptor has a growing number of putative endogenous ligands, such as tryptophan, cholesterol, and heme metabolites.

New TSPO Crystal Structures of Mutant and Heme-Bound Forms with Altered Flexibility, Ligand Binding, and Porphyrin Degradation Activity.

The ancient protein TSPO (translocator protein 18kD) is found in all kingdoms and was originally identified as a binding site of benzodiazepine drugs. Its physiological function remains unclear, although porphyrins are conserved ligands. Several crystal structures of bacterial TSPO and nuclear magnetic resonance structures of a mouse form have revealed monomer and dimer configurations, but there have been no reports of structures with a physiological ligand.

Mitochondrial respiration is controlled by Allostery, Subunit Composition and Phosphorylation Sites of Cytochrome c Oxidase: A trailblazer's tale - Bernhard Kadenbach.

In memoriam of Bernhard Kadenbach: Although the main focus of his research was the structure, function, and regulation of mitochondrial cytochrome c oxidase (CytOx), he earlier studied the mitochondrial phosphate carrier and found an essential role of cardiolipin. Later, he discovered tissue-specific and developmental-specific protein isoforms of CytOx. Defective activity of CytOx is found with increasing age in human muscle and neuronal cells resulting in mitochondrial diseases.

TSPO protein binding partners in bacteria, animals, and plants.

The ancient membrane protein TSPO is phylogenetically widespread from archaea and bacteria to insects, vertebrates, plants, and fungi. TSPO's primary amino acid sequence is only modestly conserved between diverse species, although its five transmembrane helical structure appears mainly conserved. Its cellular location and orientation in membranes have been reported to vary between species and tissues, with implications for potential diverse binding partners and function.

Highlighting membrane protein structure and function: A celebration of the Protein Data Bank.

Biological membranes define the boundaries of cells and compartmentalize the chemical and physical processes required for life. Many biological processes are carried out by proteins embedded in or associated with such membranes. Determination of membrane protein (MP) structures at atomic or near-atomic resolution plays a vital role in elucidating their structural and functional impact in biology.

Membrane-Mediated Ligand Unbinding of the PK-11195 Ligand from TSPO.

The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor, is of longstanding medical interest as both a biomarker for neuroinjury and a potential drug target for neuroinflammation and other disorders. Recently, it was shown that ligand residence time is a key factor determining steroidogenic efficacy of TSPO-binding compounds. This spurs interest in simulations of (un)binding pathways of TSPO ligands, which could reveal the molecular interactions governing ligand residence time.

Collision Induced Unfolding Classifies Ligands Bound to the Integral Membrane Translocator Protein.

Membrane proteins represent most current therapeutic targets, yet remain understudied due to their insolubility in aqueous solvents and generally low yields during purification and expression. Ion mobility-mass spectrometry and collision induced unfolding experiments have recently garnered attention as methods capable of directly detecting and quantifying ligand binding within a wide range of membrane protein systems. Despite prior success, ionized surfactant often creates chemical noise patterns resulting in significant challenges surrounding the study of small membrane protein-ligand complexes.

Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry.

Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme.

Definition of the Interaction Domain and Electron Transfer Route between Cytochrome c and Cytochrome Oxidase.

The reaction between cytochrome c () and cytochrome c oxidase (CcO) was studied using horse cytochrome c derivatives labeled with ruthenium trisbipyridine at Cys 39 (Ru-39-). Flash photolysis of a 1:1 complex between Ru-39- and bovine CcO at a low ionic strength resulted in the electron transfer from photoreduced heme c to Cu with an intracomplex rate constant of = 6 × 10 s. The K13A, K72A, K86A, and K87A Ru-39- mutants had nearly the same value but bound much more weakly to bovine CcO than wild-type Ru-39-, indicating that lysines 13, 72, 86, and 87 were involved in electrostatic binding to CcO, but were not involved in the electron transfer pathway.

The K-path entrance in cytochrome c oxidase is defined by mutation of E101 and controlled by an adjacent ligand binding domain.

Three mutant forms of Rhodobacter sphaeroides cytochrome c oxidase (RsCcO) were created to test for multiple K-path entry sites (E101W), the existence of an "upper ligand site" (M350 W), and the nature and binding specificity of the "lower ligand site" (P315W/E101A) in the region of a crystallographically-defined deoxycholate at the K-path entrance. The effects of inhibitory and stimulatory detergents (dodecyl maltoside and Tween20) on these mutants are presented, as well as competition with other ligands, including the potentially physiologically relevant ligands cholesterol and retinoic acid. Ligands are shown to be able to compete with natural lipids to affect the activity of membrane-bound RsCcO.

Role of conformational change and K-path ligands in controlling cytochrome oxidase activity.

Given the central role of cytochrome oxidase (CO) in health and disease, it is an increasingly important question as to how the activity and efficiency of this key enzyme are regulated to respond to a variety of metabolic states. The present paper summarizes evidence for two modes of regulation of activity: first, by redox-induced conformational changes involving the K-proton uptake path; and secondly, by ligand binding to a conserved site immediately adjacent to the entrance of the K-path that leads to the active site. Both these phenomena highlight the importance of the K-path in control of CO.

Translocator Protein 18 kDa (TSPO): An Old Protein with New Functions?

Translocator protein 18 kDa (TSPO) was previously known as the peripheral benzodiazepine receptor (PBR) in eukaryotes, where it is mainly localized to the mitochondrial outer membrane. Considerable evidence indicates that it plays regulatory roles in steroidogenesis and apoptosis and is involved in various human diseases, such as metastatic cancer, Alzheimer's and Parkinson's disease, inflammation, and anxiety disorders. Ligands of TSPO are widely used as diagnostic tools and treatment options, despite there being no clear understanding of the function of TSPO.

Response to Comment on "Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism".

Wang comments that the diffraction data for the structure of the A139T mutant of translocator protein TSPO from Rhodobacter sphaeroides should be used to 1.65 instead of 1.8 angstroms and that the density interpreted as porphyrin and monoolein is better fitted as polyethylene glycol.

Hypoxia Inducible Factors Modulate Mitochondrial Oxygen Consumption and Transcriptional Regulation of Nuclear-Encoded Electron Transport Chain Genes.

Hypoxia inducible factor-1 (HIF1) is a stress-responsive nuclear transcription factor that is activated with a decrease in oxygen availability. HIF1 regulates the expression of genes involved in a cell's adaptation to hypoxic stress, including those with mitochondrial specific function. To gain a more comprehensive understanding of the role of HIF1 in mitochondrial homeostasis, we studied the link between hypoxia, HIF1 transactivation, and electron transport chain (ETC) function.

Evolving understanding of translocator protein 18 kDa (TSPO).

The translocator protein 18 kDa (TSPO) has been the focus of intense research by the biomedical community and the pharmaceutical industry because of its apparent involvement in many disease-related processes. These include steroidogenesis, apoptosis, inflammation, neurological disease and cancer, resulting in the use of TSPO as a biomarker and its potential as a drug target. Despite more than 30 years of study, the precise function of TSPO remains elusive.

Identification of a key cholesterol binding enhancement motif in translocator protein 18 kDa.

Translocator protein 18 kDa (TSPO) in the mitochondrial outer membrane has been implicated in cholesterol transport regulating steroidogenesis. A human single polymorphism associated with anxiety disorders (A147T) and reduced pregnenolone production is adjacent to TSPO's cholesterol binding motif. In a mutant mimicking this polymorphism, we observe a lower level of binding of cholesterol.

Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism.

The 18-kilodalton translocator protein (TSPO), proposed to be a key player in cholesterol transport into mitochondria, is highly expressed in steroidogenic tissues, metastatic cancer, and inflammatory and neurological diseases such as Alzheimer's and Parkinson's. TSPO ligands, including benzodiazepine drugs, are implicated in regulating apoptosis and are extensively used in diagnostic imaging. We report crystal structures (at 1.

Phase-sensitive detection in modulation excitation spectroscopy applied to potential induced electron transfer in cytochrome c oxidase.

Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides was investigated by modulated excitation surface-enhanced infrared-absorption spectroscopy (SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During modulated excitation by periodic potential pulses with frequencies between 20 and 500 Hz, time-resolved infrared spectra were measured by the step-scan technique, with time resolution in the millisecond range.

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

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.

A conserved amphipathic ligand binding region influences k-path-dependent activity of cytochrome C oxidase.

A conserved, crystallographically defined bile acid binding site was originally identified in the membrane domain of mammalian and bacterial cytochrome c oxidase (CcO). Current studies show other amphipathic molecules including detergents, fatty acids, steroids, and porphyrins bind to this site and affect the already 50% inhibited activity of the E101A mutant of Rhodobacter sphaeroides CcO as well as altering the activity of wild-type and bovine enzymes. Dodecyl maltoside, Triton X100, C12E8, lysophophatidylcholine, and CHOBIMALT detergents further inhibit RsCcO E101A, with lesser inhibition observed in wild-type.

Spectral identification of intermediates generated during the reaction of dioxygen with the wild-type and EQ(I-286) mutant of Rhodobacter sphaeroides cytochrome c oxidase.

Cytochrome c oxidase from Rhodobacter sphaeroides is frequently used to model the more complex mitochondrial enzyme. The O(2) reduction in both enzymes is generally described by a unidirectional mechanism involving the sequential formation of the ferrous-oxy complex (compound A), the P(R) state, the oxyferryl F form, and the oxidized state. In this study we investigated the reaction of dioxygen with the wild-type reduced R.