RESUMEN
Lipid nanodiscs have become a standard tool for studying membrane proteins, including using single particle cryo-electron microscopy (cryo-EM). We find that reconstituting the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodiscs produces distinct structures by cryo-EM. The effect of the nanodisc on ELIC structure extends to the extracellular domain and agonist binding site. Additionally, molecular dynamic simulations indicate that nanodiscs of different size impact ELIC structure and that the nanodisc scaffold directly interacts with ELIC. These findings suggest that the nanodisc plays a crucial role in determining the structure of pLGICs, and that reconstitution of ion channels in larger nanodiscs may better approximate a lipid membrane environment.
Asunto(s)
Canales Iónicos Activados por Ligandos , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Sitios de Unión , LípidosRESUMEN
Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.
Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio de Dominio Poro en Tándem/genética , Fosfolípidos , Potenciales de la Membrana , Potasio/metabolismoRESUMEN
Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.
Asunto(s)
Canales Iónicos Activados por Ligandos , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Fosfolípidos , Sitios de Unión , Fosfatidilgliceroles , LiposomasRESUMEN
Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs) but the mechanism of inhibition is not well understood. The PUFA, docosahexaenoic acid (DHA), inhibits agonist responses of the pLGIC, ELIC, more effectively than palmitic acid, similar to the effects observed in the GABAA receptor and nicotinic acetylcholine receptor. Using photo-affinity labeling and coarse-grained molecular dynamics simulations, we identified two fatty acid binding sites in the outer transmembrane domain (TMD) of ELIC. Fatty acid binding to the photolabeled sites is selective for DHA over palmitic acid, and specific for an agonist-bound state. Hexadecyl-methanethiosulfonate modification of one of the two fatty acid binding sites in the outer TMD recapitulates the inhibitory effect of PUFAs in ELIC. The results demonstrate that DHA selectively binds to multiple sites in the outer TMD of ELIC, but that state-dependent binding to a single intrasubunit site mediates DHA inhibition of ELIC.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Canales Iónicos Activados por Ligandos/metabolismo , Sitios de Unión , Simulación de Dinámica Molecular , Dominios ProteicosRESUMEN
Photodeoxygenation of dibenzothiophene S-oxide and its derivatives have been used to generate atomic oxygen [O(3P)] to examine its effect on proteins, nucleic acids, and lipids. The unique reactivity and selectivity of O(3P) have shown distinct oxidation products and outcomes in biomolecules and cell-based studies. To understand the scope of its global impact on the cell, we treated MDA-MB-231 cells with 2,8-diacetoxymethyldibenzothiophene S-oxide and UV-A light to produce O(3P) without targeting a specific cell organelle. Cellular responses to O(3P)-release were analyzed using cell viability and cell cycle phase determination assays. Cell death was observed when cells were treated with higher concentrations of sulfoxides and UV-A light. However, significant differences in cell cycle phases due to UV-A irradiation of the sulfoxide were not observed. We further performed RNA-Seq analysis to study the underlying biological processes at play, and while UV-irradiation itself influenced gene expression, there were 9 upregulated and 8 downregulated genes that could be attributed to photodeoxygenation.
Asunto(s)
Óxidos , Tiofenos , Oxidación-Reducción , Tiofenos/farmacología , Rayos UltravioletaRESUMEN
Sulfoximines are popular scaffolds in drug discovery due to their hydrogen bonding properties and chemical stability. In recent years, the role of reactive intermediates such as nitrenes has been studied in the synthesis and degradation of sulfoximines. In this work, the photochemistry of N-phenyl dibenzothiophene sulfoximine [5-(phenylimino)-5H-5λ4 -dibenzo[b,d]thiophene S-oxide] was analyzed. The structure resembles a combination of N-phenyl iminodibenzothiophene and dibenzothiophene S-oxide, which generate nitrene and O(3 P) upon UV-A irradiation, respectively. The photochemistry of N-phenyl dibenzothiophene sulfoximine was explored by monitoring the formation of azobenzene, a photoproduct of triplet nitrene, using direct irradiation and sensitized experiments. The reactivity profile was further studied through direct irradiation experiments in the presence of diethylamine (DEA) as a nucleophile. The studies demonstrated that N-phenyl dibenzothiophene sulfoximine underwent S-N photocleavage to release singlet phenyl nitrene which formed a mixture of azepines in the presence of DEA and generated moderate amounts of azobenzene in the absence of DEA to indicate formation of triplet phenyl nitrene.
Asunto(s)
Óxidos , Tiofenos , Estructura Molecular , Fotoquímica , Tiofenos/químicaRESUMEN
Lipids modulate the function of many ion channels, possibly through direct lipid-protein interactions. The recent outpouring of ion channel structures by cryo-EM has revealed many lipid binding sites. Whether these sites mediate lipid modulation of ion channel function is not firmly established in most cases. However, it is intriguing that many of these lipid binding sites are also known sites for other allosteric modulators or drugs, supporting the notion that lipids act as endogenous allosteric modulators through these sites. Here, we review such lipid-drug binding sites, focusing on pentameric ligand-gated ion channels and transient receptor potential channels. Notable examples include sites for phospholipids and sterols that are shared by anesthetics and vanilloids. We discuss some implications of lipid binding at these sites including the possibility that lipids can alter drug potency or that understanding protein-lipid interactions can guide drug design. Structures are only the first step toward understanding the mechanism of lipid modulation at these sites. Looking forward, we identify knowledge gaps in the field and approaches to address them. These include defining the effects of lipids on channel function in reconstituted systems using asymmetric membranes and measuring lipid binding affinities at specific sites using native mass spectrometry, fluorescence binding assays, and computational approaches.
RESUMEN
Oxidation of thiols yield sulfenic acids, which are very unstable intermediates. As sulfenic acids are reactive, they form disulfides in the presence of thiols. However, sulfenic acids also oxidize to sulfinic acids (-SO2H) and sulfonic acids (-SO3H) at higher concentrations of oxidants. Hydrogen peroxide is a commonly used oxidant for the oxidation of thiols to yield sulfenic acids. However, hydrogen peroxide also oxidizes other reactive functional groups present in a molecule. In this work, the reaction intermediates arising from the oxidation of sterically hindered thiols by aryl chalcogen oxides, dibenzothiophene S-oxide (DBTO), dibenzoselenophene Se-oxide (DBSeO), and dibenzotellurophene Te-oxide (DBTeO), were investigated. Photodeoxygenation of DBTO produces triplet atomic oxygen [O(3P)], which has previously shown to preferentially react with thiols over other functional groups. Similarly, aryl selenoxides have also shown that they can thermally react selectively with thiols at room temperature to yield disulfides. Conversely, aryl telluroxides have been reported to oxidize thiols to disulfides thermally with no selectivity toward thiols. The results from this study demonstrate that sulfenic acids are an intermediate in the oxidation of thiols by DBTeO and by photodeoxygenation of DBTO. The results also showed that the oxidation of thiols by DBSeO yields sulfonic acids. Triptycene-9-thiol and 9-fluorotriptycene-10-thiol were for the thiols used in this oxidation reaction. This work expands the list of oxidants that can be used to oxidize thiols to obtain sulfenic acids.
RESUMEN
Photodeoxygenation of Dibenzothiophene-S-oxide (DBTO) in UV-A light produces atomic oxygen [O(3P)] and the corresponding sulfide, dibenzothiophene (DBT). Recently, DBTO has been derivatized to study the effect of UV-A light-driven photodeoxygenation in lipids, proteins, and nucleic acids. In this study, two DBTO derivatives with triphenylphosphonium groups were synthesized to promote mitochondrial accumulation. The sulfone analogs of these derivatives were also synthesized and used as fluorescent mitochondrial dyes to assess localization in mitochondria of HeLa cells. These derivatives were then used to study the effect of photodeoxygenation on MDA-MB-231 breast cancer cell line using cell viability assays, cell cycle phase determination tests, and RNA-Seq analysis. The DBTO derivatives were found to significantly decrease cell viability only after UV-A irradiation as a result of generating corresponding sulfides that were found to significantly affect gene expression and cell cycle.
Asunto(s)
Antineoplásicos/síntesis química , Citotoxinas/síntesis química , Compuestos Organofosforados/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Citotoxinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Compuestos Organofosforados/farmacología , Oxígeno/química , Oxígeno/metabolismo , Procesos Fotoquímicos , Especies Reactivas de Oxígeno/química , Especies Reactivas de Oxígeno/metabolismo , Tiofenos/química , Rayos UltravioletaRESUMEN
Native mass spectrometry (MS) provides the capacity to monitor membrane protein complexes and noncovalent binding of ligands and lipids to membrane proteins. The charge states produced by native MS of membrane proteins often result in gas-phase protein unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, we examined the utility of alkali metal salts as a charge-reducing agent. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, Erwinia ligand-gated ion channel (ELIC). The charge-reducing effect only occurred for membrane proteins and was detergent-dependent, being most pronounced in long polyethylene glycol (PEG)-based detergents such as C10E5 and C12E8. On the basis of these results, we propose a mechanism for alkali metal charge reduction of membrane proteins. Addition of low concentrations of alkali metals may provide an advantageous approach for charge reduction of detergent-solubilized membrane proteins by native MS.
Asunto(s)
Acetatos/química , Glutamato Deshidrogenasa/química , Proteínas de la Membrana/química , Metales Alcalinos/química , Piruvato Quinasa/química , Animales , Bovinos , Detergentes/química , Glutamato Deshidrogenasa/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Oxidación-Reducción , Piruvato Quinasa/metabolismo , Conejos , Sales (Química)/química , SolubilidadRESUMEN
A beneficial property of photogenerated reactive oxygen species (ROS) is the capability of oxidant generation within a specific location or organelle inside a cell. Dibenzothiophene S-oxide (DBTO), which is known to undergo a photodeoxygenation reaction to generate ground state atomic oxygen [O(3P)] upon irradiation, was functionalized to afford localization within the plasma membrane of cells. The photochemistry, as it relates to oxidant generation, was studied and demonstrated that the functionalized DBTO derivatives generated O(3P). Irradiation of these lipophilic O(3P)-precursors in the presence of LDL and within RAW 264.7 cells afforded several oxidized lipid products (oxLP) in the form of aldehydes. The generation of a 2-hexadecenal (2-HDEA) was markedly increased in irradiations where O(3P) was putatively produced. The substantial generation of 2-HDEA is not known to accompany the production of other ROS. These cellular irradiation experiments demonstrate the potential of inducing oxidation with O(3P) in cells.
RESUMEN
Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4',6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 and 100 µm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.
Asunto(s)
Colorantes Fluorescentes/química , Sulfonas/química , Tiofenos/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Membrana Celular/química , Células HeLa , Humanos , Microscopía Fluorescente , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.
Asunto(s)
Canales Iónicos Activados por Ligandos/metabolismo , Fosfatidilgliceroles/metabolismo , Regulación Alostérica , Análisis Mutacional de ADN , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/genética , Espectrometría de Masas , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Transmisión SinápticaRESUMEN
Ground-state atomic oxygen [O(3P)] is an oxidant whose formation in solution was proposed but never proven. Polymer nanocapsules were used to physically separate dibenzothiophene S-oxide (DBTO), a source of O(3P), from an O(3P)-accepting molecule. Irradiation of polymer nanocapsules loaded with DBTO resulted in oxidation of the O(3P)-acceptor placed outside nanocapsules. The results rule out a direct oxygen atom transfer mechanism and are consistent with freely diffusing O(3P) as the oxidant.
RESUMEN
Asymmetric dibenzothiophene S, S-dioxides (DBTOOs) were synthesized and their photophysical properties examined. Through examination, the molecules fluoresced at wavelengths between 371 and 492 nm with quantum yields of fluorescence nearing 0.59. Three of the sulfonic acid sodium salt analogues were chosen to be introduced to HeLa cells, resulting in illumination of the nucleus by fluorescent microscopy. These compounds function as nuclear stains while also affording the ability to predict the localization of the corresponding sulfoxide precursor to ground-state atomic oxygen.