RESUMO
Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.
RESUMO
Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived fromâ¯Nicotiana tabacumâ¯BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.
Assuntos
Biotecnologia , COVID-19 , Humanos , Biotecnologia/métodos , Nicotiana/metabolismo , COVID-19/metabolismo , SARS-CoV-2/metabolismo , Biossíntese de Proteínas , Anticorpos Monoclonais/metabolismo , Dissulfetos/metabolismo , Sistema Livre de Células/metabolismoRESUMO
Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE ® ', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 µL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer® CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.
RESUMO
G protein-coupled receptors (GPCR) are integral membrane proteins that regulate multiple cellular processes. To obtain insights into structural properties of GPCR and mechanism of activity, these proteins should be isolated in significant (milligram) quantities, in a pure, homogenous, and stable form. Here we describe the expression and purification of type II human cannabinoid receptor CB2, a class A GPCR, in two different types of expression hosts: in Escherichia coli and in mammalian suspension cell culture Expi293. Our method allows preparation of milligram quantities of the purified receptors suitable for a wide array of downstream applications including high-resolution structural studies and functional assays.
Assuntos
Cromatografia de Afinidade/métodos , Cristalografia por Raios X/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Receptor CB2 de Canabinoide/isolamento & purificação , Receptor CB2 de Canabinoide/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
Ketamine is an anesthetic, analgesic, and antidepressant whose secondary metabolite (2R,6R)-hydroxynorketamine (HNK) has N-methyl-d-aspartate-receptor-independent antidepressant activity in a rodent model. In humans, naltrexone attenuates its antidepressant effect, consistent with opioid pathway involvement. No detailed biophysical description is available of opioid receptor binding of ketamine or its metabolites. Using molecular dynamics simulations with free energy perturbation, we characterize the binding site and affinities of ketamine and metabolites in µ and κ opioid receptors, finding a profound effect of the protonation state. G-protein recruitment assays show that HNK is an inverse agonist, attenuated by naltrexone, in these receptors with IC50 values congruous with our simulations. Overall, our findings are consistent with opioid pathway involvement in ketamine function.
Assuntos
Ketamina , Antidepressivos/farmacologia , Depressão , Ketamina/análogos & derivados , Ketamina/farmacologia , Receptores Opioides kappaRESUMO
Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Conformação Proteica/efeitos dos fármacos , Receptor CB2 de Canabinoide/genética , Receptores de Canabinoides/genética , Canfanos/farmacologia , Cicloexanóis/farmacologia , Cisteína/genética , Humanos , Mutagênese Sítio-Dirigida , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Marcadores de SpinRESUMO
Several water-soluble variants of the human mu opioid receptor (wsMORs) have been designed and expressed, which enables the detection of opioids in the nM to pM range using biosensing platforms. The tools previously developed allowed us to investigate MOR and G-protein interactions in a lipid free system to demonstrate that the lipid bilayer might not be essential for the G-protein recognition and binding. In this study, we are able to investigate G-protein interactions with MOR by using graphene enabled technology, in a lipid free system, with a high sensitivity in a real time manner. A new wsMOR with the native C-terminus was designed, expressed and then immobilized on the surfaces of scalable graphene field effect transistor (GFET)-based biosensors, enabling the recording of wsMOR/G-protein interaction with an electronic readout. G-protein only interacts with the wsMOR in the presence of the native MOR C-terminus with a KA of 32.3±11.1 pM. The electronic readout of such interaction is highly reproducible with little variance across 50 devices in one biosensor array. For devices with receptors that do not have the native C-terminus, no significant electronic response was observed in the presence of G-protein, indicating an absence of interaction. These findings reveal that lipid environment is not essential for the G-protein interaction with MOR, however, the C-terminus of MOR is essential for G-protein recognition and high affinity binding. A system to detect MOR-G protein interaction is developed. wsMOR-G2_Cter provides a novel tool to investigate the role of C terminus in the signaling pathway.
RESUMO
Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB2, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB2 in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB2 in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.
Assuntos
Colesterol/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Escherichia coli , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Receptor CB2 de Canabinoide/isolamento & purificaçãoRESUMO
Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Escherichia coli/metabolismo , Temperatura Alta , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Receptor CB2 de Canabinoide/isolamento & purificação , Receptor CB2 de Canabinoide/metabolismo , Receptores Acoplados a Proteínas G/isolamento & purificação , Proteínas RecombinantesRESUMO
Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Corantes Fluorescentes/química , Microglia/metabolismo , Receptor CB2 de Canabinoide/análise , Animais , Células CHO , Cricetulus , Modelos Animais de Doenças , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Sondas Moleculares/química , Imagem Óptica , Sensibilidade e Especificidade , Transdução de SinaisRESUMO
BACKGROUND: The misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication. METHODS: We determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for ß-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison. RESULTS: The affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the ß-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the ß-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand. CONCLUSIONS: In conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the ß-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.
Assuntos
Analgésicos Opioides/farmacologia , Butorfanol/farmacologia , Neurônios/efeitos dos fármacos , Receptores Opioides kappa/agonistas , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Analgésicos Opioides/toxicidade , Animais , Butorfanol/química , Butorfanol/metabolismo , Butorfanol/toxicidade , Linhagem Celular Tumoral , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Neurônios/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , beta-Arrestinas/metabolismoRESUMO
G protein-coupled receptors (GPCRs) comprise a large class of integral membrane proteins involved in the regulation of a broad spectrum of physiological processes and are a major target for pharmaceutical drug development. Structural studies can help advance the rational design of novel specific pharmaceuticals that target GPCRs, but such studies require expression of significant quantities of these proteins in pure, homogenous, and sufficiently stable form. An essential precursor for these structural studies is an assessment of protein stability under experimental conditions. Here we report that solubilization of a GPCR, type II cannabinoid receptor CB2, in a Façade detergent enables radioligand thermostability assessments of this receptor with low background from nonspecific interactions with lipophilic cannabinoid ligand. Furthermore, this detergent is compatible with a [35S]GTPγS radionucleotide exchange assay measuring guanine exchange factor activity that can be applied after heat treatment to further assess receptor thermostability. We demonstrate that both assays can be utilized to determine differences in CB2 thermostability caused by mutations, detergent composition, and the presence of stabilizing ligands. We report that a constitutively active CB2 variant has higher thermostability than the WT receptor, a result that differs from a previous thermostability assessment of the analogous CB1 mutation. We conclude that both ligand-binding and activity-based assays under optimized detergent conditions can support selection of thermostable variants of experimentally demanding GPCRs.
Assuntos
Detergentes/química , Ensaio Radioligante/métodos , Receptor CB2 de Canabinoide/química , Estabilidade Enzimática , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligantes , Mutação , Ligação Proteica , Desnaturação Proteica , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , SolubilidadeRESUMO
Cannabinoid receptor type II, or CB2 , is an integral membrane protein that belongs to a large class of G-protein-coupled receptors (GPCR)s. CB2 is a part of the endocannabinoid system, which plays an important role in the regulation of immune response, inflammation, and pain. Information about the structure and function of CB2 is essential for the development of specific ligands targeting this receptor. We present here a methodology for recombinant expression of CB2 and its stable isotope labeling, purification, and reconstitution into liposomes, in preparation for its characterization by nuclear magnetic resonance (NMR). Correctly folded, functional CB2 labeled with [13 C,15 N]tryptophan or uniformly labeled with 13 C and 15 N is expressed in a medium of defined composition, under controlled aeration, pH, and temperature conditions. The receptor is purified by affinity chromatography and reconstituted into lipid bilayers in the form of proteoliposomes suitable for analysis by NMR spectroscopy. © 2019 by John Wiley & Sons, Inc.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Receptor CB2 de Canabinoide , Proteínas Recombinantes , Isótopos de Carbono/química , Cromatografia de Afinidade , Humanos , Marcação por Isótopo , Lipossomos/química , Isótopos de Nitrogênio/química , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/química , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.
Assuntos
Quelantes de Cálcio/química , Cromatografia de Afinidade/métodos , Receptor CB2 de Canabinoide/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Soluções Tampão , Cálcio/química , Escherichia coli/genética , Humanos , Lipossomos/química , Camundongos , Modelos Moleculares , Coelhos , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
Cannabinoid receptor type II (CB2) is an integral membrane protein with seven transmembrane helices that belongs to the large superfamily of rhodopsin-like G protein-coupled receptors. The CB2 is a part of the endocannabinoid system that plays a vital role in regulation of immune response, inflammation, pain, and other metabolic processes. Information about the structure and function of CB2 in cell membranes is essential for development of specific pharmaceuticals that target CB2 signaling. Methodology for recombinant expression, stable isotope labeling, purification, reconstitution into liposomes, and NMR characterization of functionally active CB2 is presented. Uniformly 13C-, 15N-labeled CB2 protein is expressed by fermentation of Escherichia coli in a medium of defined composition under controlled aeration, pH, and temperature and purified by tandem affinity chromatography. The receptor reconstituted into lipid bilayers is suitable for structural studies by solid-state NMR spectroscopy.
Assuntos
Receptor CB2 de Canabinoide/química , Cromatografia de Afinidade , Meios de Cultura , Escherichia coli , Fermentação , Expressão Gênica , Humanos , Marcação por Isótopo , Ressonância Magnética Nuclear Biomolecular , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Human cannabinoid receptor CB2 belongs to the class A of G protein-coupled receptor (GPCR). CB2 is predominantly expressed in membranes of cells of immune origin and is implicated in regulation of metabolic pathways of inflammation, neurodegenerative disorders and pain sensing. High resolution structural studies of CB2 require milligram quantities of purified, structurally intact protein. While we previously reported on the methodology for expression of the recombinant CB2 and its stabilization in a functional state, here we describe an efficient protocol for purification of this protein using the Twin-Strep-tag/Strep-Tactin XT system. To improve the affinity of interaction of the recombinant CB2 with the resin, the double repeat of the Strep-tag (a sequence of eight amino acids WSHPQFEK), named the Twin-Strep-tag was attached either to the N- or C-terminus of CB2 via a short linker, and the recombinant protein was expressed in cytoplasmic membranes of E. coli as a fusion with the N-terminal maltose binding protein (MBP). The CB2 was isolated at high purity from dilute solutions containing high concentrations of detergents, glycerol and salts, by capturing onto the Strep-Tactin XT resin, and was eluted from the resin under mild conditions upon addition of biotin. Surface plasmon resonance studies performed on the purified protein demonstrate the high affinity of interaction between the Twin-Strep-tag fused to the CB2 and Strep-Tactin XT with an estimated Kd in the low nanomolar range. The affinity of binding did not vary significantly in response to the position of the tag at either N- or C-termini of the fusion. The binding capacity of the resin was several-fold higher for the tag located at the N-terminus of the protein as opposed to the C-terminus- or middle of the fusion. The variation in the length of the linker between the double repeats of the Strep-tag from 6 to 12 amino acid residues did not significantly affect the binding. The novel purification protocol reported here enables efficient isolation of a recombinant GPCR expressed at low titers in host cells. This procedure is suitable for preparation of milligram quantities of stable isotope-labelled receptor for high-resolution NMR studies.
Assuntos
Cromatografia de Afinidade/métodos , Expressão Gênica , Receptor CB2 de Canabinoide , Proteínas Recombinantes de Fusão , Escherichia coli , Humanos , Ressonância Magnética Nuclear Biomolecular , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/química , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ressonância de Plasmônio de SuperfícieRESUMO
Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor.
Assuntos
Cromatografia de Afinidade/métodos , Biologia Molecular/métodos , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/isolamento & purificação , Sequência de Aminoácidos , Escherichia coli , Histidina/química , Humanos , Oligopeptídeos/química , Receptor CB2 de Canabinoide/químicaRESUMO
BACKGROUND: Although dezocine is a partial µ-opioid receptor agonist, it is not a controlled substance. Thus, the characterization of the molecular targets of dezocine is critical for scientific and clinical implications. The goal of this study is to characterize molecular targets for dezocine and determine their implications. METHODS: A binding screen for dezocine was performed on 44 available receptors and transporter proteins. Functional assays for the novel targets were performed along with computation calculations to locate the binding site. A G protein activation study was performed for the human κ opioid receptor to determine whether dezocine is a κ-antagonist. Data are presented as mean ± standard error. RESULTS: The affinities for dezocine were 3.7 ± 0.7 nM for the µ receptor, 527 ± 70 nM for the δ-receptor, and 31.9 ± 1.9 nM for the κ-receptor. Dezocine failed to induce G protein activation with κ-opioid receptor and concentration dependently inhibited κ-agonist (salvinorin A and nalbuphine)-induced receptor activation, indicating that dezocine is a κ-antagonist. Two novel molecular targets (norepinephrine transporter and serotonin transporter) were identified. Dezocine concentration-dependently inhibited norepinephrine and serotonin reuptake in vitro. The half maximal inhibitory concentrations (expressed as pIC50) were 5.68 ± 0.11 for norepinephrine transporter and 5.86 ± 0.17 for serotonin transporter. Dezocine occupied the binding site for known norepinephrine transporter and serotonin transporter inhibitors. CONCLUSIONS: The unique molecular pharmacological profile of dezocine as a partial µ-receptor agonist, a κ-receptor antagonist, and a norepinephrine and serotonin reuptake inhibitor (via norepinephrine transporter and serotonin transporter) was revealed. These discoveries reveal potentially important novel clinical implications and drug interactions of dezocine.
Assuntos
Analgésicos Opioides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Antagonistas de Entorpecentes , Receptores Opioides/agonistas , Tetra-Hidronaftalenos/farmacologia , Humanos , Técnicas In Vitro , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
The global fold of human cannabinoid type 2 (CB2 ) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state (13)C- and (15)N magic-angle spinning (MAS) NMR, in combination with chemical-shift prediction from a structural model of the receptor obtained by microsecond-long molecular dynamics (MD) simulations. Uniformly (13)C- and (15)N-labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. (13)C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cß, and C=O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the (13) C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥ 1.5 ppm for carbons and ≥ 5.0 ppm for nitrogens). Simulated two-dimensional (13) Cα(i)-(13)C=O(i) and (13)C=O(i)-(15)NH(i + 1) dipolar-interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid-state MAS NMR.
Assuntos
Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Receptor CB2 de Canabinoide/química , Receptor CB2 de Canabinoide/metabolismo , Isótopos de Carbono/química , Escherichia coli , Humanos , Lipossomos , Isótopos de Nitrogênio/química , Dobramento de Proteína , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2.