RESUMO
Irisin is secreted by muscle, increases with exercise, and mediates certain favorable effects of physical activity. In particular, irisin has been shown to have beneficial effects in adipose tissues, brain, and bone. However, the skeletal response to exercise is less clear, and the receptor for irisin has not been identified. Here we show that irisin binds to proteins of the αV class of integrins, and biophysical studies identify interacting surfaces between irisin and αV/ß5 integrin. Chemical inhibition of the αV integrins blocks signaling and function by irisin in osteocytes and fat cells. Irisin increases both osteocytic survival and production of sclerostin, a local modulator of bone remodeling. Genetic ablation of FNDC5 (or irisin) completely blocks osteocytic osteolysis induced by ovariectomy, preventing bone loss and supporting an important role of irisin in skeletal remodeling. Identification of the irisin receptor should greatly facilitate our understanding of irisin's function in exercise and human health.
Assuntos
Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Remodelação Óssea , Fibronectinas/metabolismo , Integrina alfaV/metabolismo , Osteócitos/metabolismo , Osteólise/metabolismo , Adipócitos/patologia , Animais , Linhagem Celular Tumoral , Feminino , Fibronectinas/genética , Células HEK293 , Humanos , Integrina alfaV/genética , Camundongos , Osteócitos/patologia , Osteólise/genéticaRESUMO
The RNA sensor MDA5 recruits the signaling adaptor MAVS to initiate type I interferon signaling and downstream antiviral responses, a process that requires K63-linked polyubiquitin chains. Here, we examined the mechanisms whereby K63-polyUb chain regulate MDA5 activation. Only long unanchored K63-polyUbn (n ≥ 8) could mediate tetramerization of the caspase activation and recruitment domains of MDA5 (MDA5CARDs). Cryoelectron microscopy structures of a polyUb13-bound MDA5CARDs tetramer and a polyUb11-bound MDA5CARDs-MAVSCARD assembly revealed a tower-like formation, wherein eight Ubs tethered along the outer rim of the helical shell, bridging MDA5CARDs and MAVSCARD tetramers into proximity. ATP binding and hydrolysis promoted the stabilization of RNA-bound MDA5 prior to MAVS activation via allosteric effects on CARDs-polyUb complex. Abundant ATP prevented basal activation of apo MDA5. Our findings reveal the ordered assembly of a MDA5 signaling complex competent to recruit and activate MAVS and highlight differences with RIG-I in terms of CARD orientation and Ub sensing that suggest different abilities to induce antiviral responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Microscopia Crioeletrônica , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/ultraestrutura , Poliubiquitina/química , Poliubiquitina/metabolismo , Ligação ProteicaRESUMO
The innate immune receptor RIG-I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG-I's C-terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non-specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG-I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs-Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD-chosen RNAs to bind the helicase domain, while at the same time blocking non-specific RNAs. These findings also indicate that CHL could represent a novel target for RIG-I-based therapeutics.
Assuntos
RNA Helicases DEAD-box , RNA de Cadeia Dupla , Proteína DEAD-box 58/genética , Proteína DEAD-box 58/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Imunidade Inata , Estrutura Terciária de Proteína , RNA Viral/genéticaRESUMO
Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cristalografia por Raios X , Feminino , Humanos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Dados , Concentração de Íons de HidrogênioRESUMO
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
Assuntos
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Lasers , Camundongos , Modelos Moleculares , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Transdução de Sinais , Raios XRESUMO
Despite the growing evidence suggesting that long noncoding RNAs (lncRNAs) are critical regulators of several biological processes, their functions in the nervous system remain elusive. We have identified an lncRNA, GM12371, in hippocampal neurons that is enriched in the nucleus and necessary for synaptic communication, synapse density, synapse morphology, and dendritic tree complexity. Mechanistically, GM12371 regulates the expression of several genes involved in neuronal development and differentiation, as well as expression of specific lncRNAs and their cognate mRNA targets. Furthermore, we find that cAMP-PKA signaling up-regulates the expression of GM12371 and that its expression is essential for the activity-dependent changes in synaptic transmission in hippocampal neurons. Taken together, our data establish a key role for GM12371 in regulating synapse function.
Assuntos
Regulação da Expressão Gênica/genética , RNA Longo não Codificante/genética , Sinapses/genética , Transcrição Gênica/genética , Animais , Diferenciação Celular/genética , Feminino , Hipocampo/fisiologia , Camundongos , Neurônios/fisiologia , Gravidez , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
Hydrogen/Deuterium Exchange (HDX) coupled with Mass Spectrometry (HDX-MS) is a sensitive and robust method to probe protein conformational changes and protein-ligand interactions. HDX-MS relies on successful proteolytic digestion of target proteins under acidic conditions to localize perturbations in exchange behavior to protein structure. The ability of the protease to produce small peptides and overlapping fragments and provide sufficient coverage of the protein sequence is essential for localizing regions of interest. While the acid protease pepsin has been the enzyme of choice for HDX-MS studies, recently, it was shown that aspartic proteases from carnivorous pitcher plants of the genus Nepenthes are active under low-pH conditions and cleave at basic residues that are "forbidden" in peptic digests. In this report, we describe the utility of one of these enzymes, Nepenthesin II (NepII), in a HDX-MS workflow. A systematic and statistical analysis of data from 11 proteins (6391 amino acid residues) digested with immobilized porcine pepsin or NepII under conditions compatible with HDX-MS was performed to examine protease cleavage specificities. The cleavage of pepsin was most influenced by the amino acid residue at position P1. Phe, Leu, and Met are favored residues, each with a cleavage probability of greater than 40%. His, Lys, Arg, or Pro residues prohibit cleavage when found at the P1 position. In contrast, NepII offers advantageous cleavage to all basic residues and produces shortened peptides that could improve the spatial resolution in HDX-MS studies.
Assuntos
Enzimas Imobilizadas/química , Pepsina A/química , Proteólise , Animais , Biocatálise , Deutério/química , Medição da Troca de Deutério , Espectrometria de Massas , Sarraceniaceae/enzimologia , Especificidade por Substrato , SuínosRESUMO
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Análise de Variância , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Deutério/análise , Medição da Troca de Deutério/instrumentação , Hidrogênio/análise , Ligantes , Espectrometria de Massas/instrumentação , Peptídeos/análise , Proteínas/química , Receptores de Calcitriol/química , Reprodutibilidade dos TestesRESUMO
Liver receptor homologue-1 (LRH1) is an orphan nuclear receptor that has been shown to play a role in the transcriptional regulation of pathways involved in cancer. Elucidating the components of the LRH1 transcriptional complex to better understand endogenous regulation of the receptor as well as its role in cancer remains a high priority. A sub-cellular enrichment strategy coupled with proteomic approaches was employed to identify putative LRH1 co-regulators. Nuclear fractionation protocol was essential for detection of LRH1 peptides by mass spectrometry (MS), with most peptides being observed in the insoluble fraction (receptor bound to DNA). SERBP1 and ILF3 were identified as LRH1 interacting partners by both Western blot and MS/MS analysis. Receptor knockdown by siRNA showed an increase in SERBP1 expression, while ILF3 expression was unchanged. In contrast, receptor overexpression decreased only SERBP1 mRNA levels. Consistent with these data, in a promoter:reporter assay, binding of LRH1 to the promoter region of SERBP1 resulted in a decrease in the expression level of the reporter gene, subsequently inhibiting transcription. Given the receptor's role in cancer progression, the study here elucidates additional transcriptional machinery involved in LRH1 signaling and potentially provides new targets for therapeutics development.
Assuntos
Regulação da Expressão Gênica , Peptídeos/análise , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Fracionamento Químico , Células HEK293 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas do Fator Nuclear 90/genética , Proteínas do Fator Nuclear 90/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.
Assuntos
Medição da Troca de Deutério , Transcriptase Reversa do HIV/análise , Transcriptase Reversa do HIV/química , Espectrometria de Massas , Isótopos de NitrogênioRESUMO
Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Proteínas/química , Animais , Hormônios/química , Humanos , Hidrogênio/química , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores Citoplasmáticos e Nucleares/químicaRESUMO
Hydrogen/deuterium exchange (HDX) mass spectrometry has been widely applied to the characterization of protein dynamics. More recently, differential HDX has been shown to be effective for the characterization of ligand binding. Previously we have described a fully automated HDX system for use as a ligand screening platform. Here we describe and validate the required data analysis workflow to facilitate the use of HDX as a robust approach for ligand screening. Following acquisition of HDX data at a single on-exchange time point (n ≥ 3), one way analysis of variance in conjunction with the Tukey multiple comparison procedure is used to establish the significance of any measured difference. Analysis results are graphed with respect to a single peptide, ligand or group of ligands, or displayed as an overview within a heat map. For the heat map display, only Δ%D values with a Tukey-adjusted P value less than 0.05 are colored. Hierarchical clustering is used to bin compounds with highly similar HDX signatures. The workflow is evaluated with a small data set showing the ligand binding domain (LDB) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) screened against 10 functionally selective ligands. More significantly, data for the vitamin D receptor (VDR) in complex with 87 ligands are presented. To highlight the robustness and precision of our automated HDX platform we analyzed the data from 4191 replicate HDX measurements acquired over an eight month timeframe. Ninety six percent of these measurements were within 10 percent of the mean value. Work has begun to integrate these analysis and graphing components within our HDX software suite.
RESUMO
To examine the molecular details of ligand activation of G-protein coupled receptors (GPCRs), emphasis has been placed on structure determination of these receptors with stabilizing ligands. Here we present the methodology for receptor dynamics characterization of the GPCR human beta(2) adrenergic receptor bound to the inverse agonist carazolol using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS). The HDX MS profile of receptor bound to carazolol is consistent with thermal parameter observations in the crystal structure and provides additional information in highly dynamic regions of the receptor and chemical modifications demonstrating the highly complementary nature of the techniques. After optimization of HDX experimental conditions for this membrane protein, better than 89% sequence coverage was obtained for the receptor. The methodology presented paves the way for future analysis of beta(2)AR bound to pharmacologically distinct ligands as well as analysis of other GPCR family members.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2 , Sequência de Aminoácidos , Deutério/química , Humanos , Hidrogênio/química , Dados de Sequência Molecular , Propanolaminas/química , Estrutura Terciária de ProteínaRESUMO
The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2'-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with 15N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2'-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer's ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2'-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.
RESUMO
Here we present the use of hydrogen-deuterium exchange (HDX) mass spectrometry in analyzing the estrogen receptor beta ligand binding domain (ERbeta LBD) in the absence and presence of a variety of chemical compounds with different binding modes and pharmacological properties. Previously, we reported the use of HDX as a method for predicting the tissue selectivity of ERalpha ligands. HDX profiles of ERalpha LBD in complex with ligand could differentiate compounds of the same chemotype. In contrast, similar analysis of ERbeta LBD showed correlation to the compound chemical structures but little correlation with compound tissue selectivity. The different HDX patterns observed for ERbeta LBD when compared to those for ERalpha LBD bound to the same chemical compounds serve as an indication that ERbeta LBD undergoes a different structural response to the same ligand when compared to ERalpha LBD. The conformational dynamics revealed by HDX for ERbeta LBD together with those for ERalpha LBD shed light on ER ligand interactions and offer new structural insights. The compound-specific perturbations in HDX kinetics observed for each of the two isoforms should aid the development of subtype-selective ER ligands.
Assuntos
Medição da Troca de Deutério , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Linhagem Celular , Cristalografia por Raios X , Medição da Troca de Deutério/métodos , Estradiol/metabolismo , Receptor alfa de Estrogênio/química , Receptor beta de Estrogênio/química , Genisteína/metabolismo , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismoRESUMO
Proteins are not rigid bodies under their physiological conditions. Here we discuss a solution-phase structural proteomics technique, hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS), as a means to study protein dynamics, which can complement other structural approaches. We outline the background theory and highlight the utility of HDX-MS measurements in two case studies involving a nuclear receptor and an innate immunity receptor. We also discuss emerging software advances for improving data analysis and three-dimensional visualization.
Assuntos
Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Proteínas/química , Proteínas/metabolismo , Cinética , Conformação ProteicaRESUMO
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a biophysical technique well suited to the characterization of protein dynamics and protein-ligand interactions. In order to accurately define the rate of exchange, HDX experiments require the repeated measure of deuterium incorporation into the target protein across a range of time points. Accordingly, the HDX-MS experiment is well suited to automation, and a number of automated systems for HDX-MS have been developed. The most widely utilized platforms all operate an integrated design, where robotic liquid handling is interfaced directly with a mass spectrometer. With integrated designs, the exchange samples are prepared and injected into the LC-MS following a "real-time" serial workflow. Here we describe a new HDX-MS platform that is comprised of two complementary pieces of automation that disconnect the sample preparation from the LC-MS analysis. For preparation, a plate-based automation system is used to prepare samples in parallel, followed by immediate freezing and storage. A second piece of automation has been constructed to perform the thawing and LC-MS analysis of frozen samples in a serial mode and has been optimized to maximize the duty cycle of the mass spectrometer. The decoupled configuration described here reduces experiment time, significantly improves capacity, and improves the flexibility of the platform when compared with a fully integrated system.
Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Descoberta de Drogas/economia , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Desenho de Equipamento , Análise de Injeção de Fluxo/economia , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério/economia , Espectrometria de Massa com Troca Hidrogênio-Deutério/instrumentação , Ligantes , Proteínas/químicaRESUMO
Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.