RESUMO
Cooperative interactions in protein-protein interfaces demonstrate the interdependency or the linked network-like behavior and their effect on the coupling of proteins. Cooperative interactions also could cause ripple or allosteric effects at a distance in protein-protein interfaces. Although they are critically important in protein-protein interfaces, it is challenging to determine which amino acid pair interactions are cooperative. In this work, we have used Bayesian network modeling, an interpretable machine learning method, combined with molecular dynamics trajectories to identify the residue pairs that show high cooperativity and their allosteric effect in the interface of G protein-coupled receptor (GPCR) complexes with Gα subunits. Our results reveal six GPCR:Gα contacts that are common to the different Gα subtypes and show strong cooperativity in the formation of interface. Both the C terminus helix5 and the core of the G protein are codependent entities and play an important role in GPCR coupling. We show that a promiscuous GPCR coupling to different Gα subtypes, makes all the GPCR:Gα contacts that are specific to each Gα subtype (Gαs, Gαi, and Gαq). This work underscores the potential of data-driven Bayesian network modeling in elucidating the intricate dependencies and selectivity determinants in GPCR:G protein complexes, offering valuable insights into the dynamic nature of these essential cellular signaling components.
Assuntos
Teorema de Bayes , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genéticaRESUMO
Heterotrimeric GTP-binding protein alpha subunit (Gα) and its cognate regulator of G-protein signaling (RGS) protein transduce signals in eukaryotes spanning protists, amoeba, animals, fungi, and plants. The core catalytic mechanisms of the GTPase activity of Gα and the interaction interface with RGS for the acceleration of GTP hydrolysis seem to be conserved across these groups; however, the RGS gene is under low selective pressure in plants, resulting in its frequent loss. Our current understanding of the structural basis of Gα:RGS regulation in plants has been shaped by Arabidopsis Gα, (AtGPA1), which has a cognate RGS protein. To gain a comprehensive understanding of this regulation beyond Arabidopsis, we obtained the x-ray crystal structures of Oryza sativa Gα, which has no RGS, and Selaginella moellendorffi (a lycophyte) Gα that has low sequence similarity with AtGPA1 but has an RGS. We show that the three-dimensional structure, protein-protein interaction with RGS, and the dynamic features of these Gα are similar to AtGPA1 and metazoan Gα. Molecular dynamic simulation of the Gα-RGS interaction identifies the contacts established by specific residues of the switch regions of GTP-bound Gα, crucial for this interaction, but finds no significant difference due to specific amino acid substitutions. Together, our data provide valuable insights into the regulatory mechanisms of plant G-proteins but do not support the hypothesis of adaptive co-evolution of Gα:RGS proteins in plants.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP , Modelos Moleculares , Proteínas de Plantas , Proteínas RGS , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Oryza/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , Proteínas RGS/metabolismo , Proteínas RGS/química , Proteínas RGS/genética , Relação Estrutura-Atividade , Selaginellaceae/genética , Selaginellaceae/metabolismo , Estrutura Quaternária de ProteínaRESUMO
Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (Gsα) alone, in the intact Gsαß1γ2 heterotrimer or in complex with membrane-embedded human adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the ßγ subunit, the lipid bilayer and A2AR. The α1 helix of Gsα exhibits significant intermediate timescale dynamics. The α4ß6 loop and α5 helix undergo membrane/receptor interactions and order-disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the ßγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Proteínas Heterotriméricas de Ligação ao GTP , Humanos , Modelos Moleculares , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Conformação Proteica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Ligação ProteicaRESUMO
The Gα subunit is an important component of the heterotrimeric G-protein complex and an integral component of several signal transduction pathways. It plays crucial roles in the diverse processes of plant growth and development, including the response to abiotic stress, regulation of root development, involvement in stomatal movement, and participation in hormone responses, which have been well investigated in many species. However, no comprehensive analysis has identified and explored the evolution, expression pattern characteristics and heat stress response of the Gα subunit genes in Rosaceae. In this study, 52 Gα subunit genes were identified in eight Rosaceae species; these genes were divided into three subfamilies (I, II, and III) based on their phylogenetic, conserved motif, and structural characteristics. Whole genome and dispersed duplication events were found to have contributed significantly to the expansion of the Gα subunit gene family, and purifying selection to have played a key role in the evolution of Gα subunit genes. An expression analysis identified some PbrGPA genes that were highly expressed in leaf, root, and fruit, and exhibited diverse spatiotemporal expression models in pear. Under abiotic stress conditions, the mRNA transcript levels of PbrGPA genes were up-regulated in response to high temperature treatment in leaves. Furthermore, three Gα subunit genes were shown to be located in the plasma membrane and nucleus in pear. In conclusion, the study of the Gα subunit gene family will help us to better understand its evolutionary history and expression patterns, while facilitating further investigations into the function of the Gα subunit gene in response to heat stress.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Rosaceae/genética , Rosaceae/fisiologia , Motivos de Aminoácidos , Evolução Molecular , Subunidades alfa de Proteínas de Ligação ao GTP/química , Duplicação Gênica , Expressão Gênica , Genoma de Planta , Resposta ao Choque Térmico , Filogenia , Pyrus/genética , Pyrus/fisiologia , Rosaceae/classificaçãoRESUMO
Classical pharmacological models have incorporated an "intrinsic efficacy" parameter to capture system-independent effects of G protein-coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand "molecular efficacy" that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor-G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor-G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor-G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Receptores Adrenérgicos beta 2/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Ligação ProteicaRESUMO
G-protein coupled receptors (GPCRs) are among the most versatile signal transducers in the cell. Once activated, GPCRs sample a large conformational space and couple to G-proteins to initiate distinct signaling pathways. The dynamical behavior of GPCR-G-protein complexes is difficult characterize structurally, and it might hinder obtaining routine high-resolution density maps in single-particle reconstructions. Here, we used variability analysis on the rhodopsin-Gi-Fab16 complex cryo-EM dataset, and the results provide insights into the dynamic nature of the receptor-complex interaction. We compare the outcome of this analysis with recent results obtained on the cannabinoid-Gi- and secretin-Gs-receptor complexes. Despite differences related to the biochemical compositions of the three samples, a set of consensus movements emerges. We anticipate that systematic variability analysis on GPCR-G-protein complexes may provide useful information not only at the biological level, but also for improving the preparation of more stable samples for cryo-EM single-particle analysis.
Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/química , Complexos Multiproteicos/química , Receptores Acoplados a Proteínas G/química , Bases de Dados de Proteínas , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Imageamento Tridimensional , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Complexos Multiproteicos/metabolismo , Conformação Proteica em alfa-Hélice , Receptores Acoplados a Proteínas G/metabolismo , Rodopsina/química , Rodopsina/metabolismoRESUMO
Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-Gi complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMOD384R and SMOG111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.
Assuntos
Colesterol/análogos & derivados , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Colesterol/química , Colesterol/metabolismo , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única , Receptor Smoothened/agonistas , Receptor Smoothened/química , Receptor Smoothened/genética , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Subunidades alfa de Proteínas de Ligação ao GTP/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina/ultraestrutura , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/ultraestrutura , Fosforilação , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Controle de QualidadeRESUMO
Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Sítios de Ligação , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Camundongos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Eukaryotic heterotrimeric guanine nucleotide-binding proteins consist of α, ß, and γ subunits, which act as molecular switches to regulate a number of fundamental cellular processes. In the oomycete pathogen Phytophthora sojae, the sole G protein α subunit (Gα; encoded by PsGPA1) has been found to be involved in zoospore mobility and virulence, but how it functions remains unclear. In this study, we show that the Gα subunit PsGPA1 directly interacts with PsYPK1, a serine/threonine protein kinase that consists of an N-terminal region with unknown function and a C-terminal region with a conserved catalytic kinase domain. We generated knockout and knockout-complemented strains of PsYPK1 and found that deletion of PsYPK1 resulted in a pronounced reduction in the production of sporangia and oospores, in mycelial growth on nutrient poor medium, and in virulence. PsYPK1 exhibits a cytoplasmic-nuclear localization pattern that is essential for sporangium formation and virulence of P. sojae. Interestingly, PsGPA1 overexpression was found to prevent nuclear localization of PsYPK1 by exclusively binding to the N-terminal region of PsYPK1, therefore accounting for its negative role in sporangium formation. Our data demonstrate that PsGPA1 negatively regulates sporangium formation by repressing the nuclear localization of its downstream kinase PsYPK1.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Phytophthora/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Esporos/crescimento & desenvolvimento , Núcleo Celular/genética , Núcleo Celular/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Phytophthora/genética , Phytophthora/crescimento & desenvolvimento , Phytophthora/patogenicidade , Doenças das Plantas/parasitologia , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Glycine max/parasitologia , Esporos/enzimologia , Esporos/genética , Esporos/metabolismo , VirulênciaRESUMO
Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Gα hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Gα proteins. Here, we examined the structure of the complex of Ric8A with minimized Gαi (miniGαi) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGαi complex by steered molecular dynamics (SMD) simulations. A small set of models of the complex featured minimal clash scores, excellent agreement with the experimental SAXS data, and a large-scale rearrangement of the signal-transducing α5-helix of Gα away from its ß-sheet core. The resulting interface involved the Gα α5-helix bound to the concave surface of Ric8A and the Gα ß-sheet that wraps around the C-terminal part of the Ric8A armadillo domain, leading to a severe disruption of the GDP-binding site. Further modeling of the flexible C-terminal tail of Ric8A indicated that it interacts with the effector surface of Gα. This smaller interface may enable the Ric8A-bound Gα to interact with GTP. The two-interface interaction with Gα described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Bovinos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Eletricidade Estática , Difração de Raios XRESUMO
Aromatic side chains in proteins are often directly evolved in stabilizing the hydrophobic core, protein binding, or enzymatic activity. They are also responsible for specific local dynamic processes, such as histidine tautomerization or ring flips. Despite their importance, they are often not targeted directly by NMR spectroscopy, because of spectroscopic complications and challenges. This chapter addresses state-of-the-art site-selective 13C-labeling methods for aromatic side chains, and describes how they solve several of the spectroscopic issues. A special emphasis is put on thereby enabled protein dynamics experiments of aromatic side chains.
Assuntos
Aminoácidos Aromáticos/química , Isótopos de Carbono/química , Subunidades alfa de Proteínas de Ligação ao GTP/química , Glucose/química , Marcação por Isótopo/métodos , Espectroscopia de Ressonância Magnética/métodos , Coloração e Rotulagem/métodos , Deutério/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética/instrumentação , Simulação de Dinâmica Molecular , Proteínas Recombinantes/química , TermodinâmicaRESUMO
G proteins regulate shoot, root, and epidermis development, as well as biotic stress tolerance in plants; however, most studies have examined model plants and less attention has been paid to the function of G proteins in horticultural plants. Here, we identified a G protein, CsGPA1, from cucumber and studied its function in seedling development of cucumbers. CsGPA1 is a peptide of 392 amino acids with a predicted molecular mass of 44.6 kDa. Spatiotemporal expression analysis found that endogenous CsGPA1 was highly expressed in roots and young leaves. Immunohistochemical assay revealed that functional CsGPA1 was present in the plasma membranes of the epidermis and cortex, and in the cytosol of the endodermis, parenchyma, and vasculature of root meristematic cells. In comparison with wild-type seedlings, CsGPA1-overexpressing transgenic lines exhibited enhanced seed germination and earlier seedling development including hypocotyl elongation and root growth. In contrast, RNAi silencing of the CsGPA1 gene inhibited seedling growth and development. Further study showed that CsGPA1 controled hypocotyl elongation and root growth via promoting cell size and the meristem of hypocotyl and root tip cells of cucumber plants. Our study expands the roles of G proteins in plants and helps to elucidate the mechanism by which cucumber regulates early seedling development.
Assuntos
Cucumis sativus/crescimento & desenvolvimento , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Hipocótilo/crescimento & desenvolvimento , Raízes de Plantas/crescimento & desenvolvimento , Membrana Celular/química , Citosol/química , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Imuno-Histoquímica , Peso Molecular , Plântula/crescimento & desenvolvimento , Análise Espaço-TemporalRESUMO
Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein-coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif-containing proteins.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfolipase C delta/química , Fosfolipase C delta/genética , Motivos de Aminoácidos , Cristalografia por Raios X , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Fosfolipase C delta/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by mediating a GDP to GTP exchange in the Gα subunit. This leads to dissociation of the heterotrimer into Gα-GTP and Gßγ dimer. The Gα-GTP and Gßγ dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated Gßγ-signaling is a central element of various neurological and cancer-related anomalies. However, Gßγ also serves as a negative regulator of Gα that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Here we report a llama-derived nanobody (Nb5) that binds tightly to the Gßγ dimer. Nb5 responds to all combinations of ß-subtypes and γ-subtypes and competes with other Gßγ-regulatory proteins for a common binding site on the Gßγ dimer. Despite its inhibitory effect on Gßγ-mediated signaling, Nb5 has no effect on Gαq-mediated and Gαs-mediated signaling events in living cells.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Anticorpos de Domínio Único/metabolismo , Sítios de Ligação , Dimerização , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Guanosina Trifosfato/metabolismo , Humanos , Ligação Proteica , Transdução de Sinais , Anticorpos de Domínio Único/químicaRESUMO
Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser435 and Thr440 in rat Ric-8A (corresponding to Ser436 and Thr441 in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser435 and Thr440 are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser468 and Ser473, were also phosphorylated. Mutation of the genomic copy of ric-8 in Caenorhabditis elegans to encode alanine in the homologous sites resulted in characteristic ric-8 reduction-of-function phenotypes that are associated with defective Gq and Gs signaling, including reduced locomotion and defective egg laying. The C. elegans ric-8 phosphorylation site mutant phenotypes were partially rescued by chemical stimulation of Gq signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Conformação Proteica , Ratos , Serina/química , Serina/genética , Serina/metabolismo , Transdução de Sinais , Treonina/química , Treonina/genética , Treonina/metabolismoRESUMO
BACKGROUND: Heterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gßγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete. RESULTS: We have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events. CONCLUSIONS: Our curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein - GPCR signaling pathways which radiated in Vertebrata evolution.
Assuntos
Evolução Molecular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Vertebrados/genética , Animais , Proteínas de Ligação a DNA/genética , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Duplicação Gênica , Motivos de Nucleotídeos , Filogenia , Retroelementos , Transdução de Sinais , Vertebrados/classificaçãoRESUMO
Gnaq, one of Guanine nucleotide-binding protein α subunits, was isolated from cellular nucleus extracts of oyster Crassostrea hongkongensis gills with biotin-labeled ChHsc70 promoter by means of DNA-affinity purification, and preliminarily identified with mass spectrometry analysis. ChGnaq mRNA depletion by RNAi technique led to clear reduction in ChHsc70 mRNA expression of C. hongkongensis hemocytes. Correspondently, ChGnaq over-expression in heterologous HEK293T cells correlated with elevated expression activation of ChHsc70 promoter. Quantitative real time PCR analysis showed that both ChHsc70 and ChGnaq transcriptions were responsive to external physical/chemical stresses by heat, CdCl2 and NP. This suggested a plausible association between ChHsc70 and ChGnaq in the stress-induced genetic regulatory pathway. This study discovered a positively regulatory role of ChGnaq in controlling ChHsc70 transcription of C. hongkongensis, and conduced to a better understanding of the regulatory mechanisms in control of Hsc70 transcription.
Assuntos
Crassostrea/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Choque Térmico HSC70/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Biotina/metabolismo , Cloreto de Cádmio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas de Choque Térmico HSC70/metabolismo , Hemócitos/efeitos dos fármacos , Hemócitos/metabolismo , Temperatura Alta , Humanos , Luciferases/metabolismo , Fenóis/farmacologia , Regiões Promotoras Genéticas , Coloração e Rotulagem , Transcrição Gênica/efeitos dos fármacosRESUMO
G-protein subunit α-11 (Gα11 ) couples the calcium-sensing receptor (CaSR) to phospholipase C (PLC)-mediated intracellular calcium (Ca2+i ) and mitogen-activated protein kinase (MAPK) signaling, which in the parathyroid glands and kidneys regulates parathyroid hormone release and urinary calcium excretion, respectively. Heterozygous germline loss-of-function Gα11 mutations cause familial hypocalciuric hypercalcemia type 2 (FHH2), for which effective therapies are currently not available. Here, we report a novel heterozygous Gα11 germline mutation, Phe220Ser, which was associated with hypercalcemia in a family with FHH2. Homology modeling showed the wild-type (WT) Phe220 nonpolar residue to form part of a cluster of hydrophobic residues within a highly conserved cleft region of Gα11 , which binds to and activates PLC; and predicted that substitution of Phe220 with the mutant Ser220 polar hydrophilic residue would disrupt PLC-mediated signaling. In vitro studies involving transient transfection of WT and mutant Gα11 proteins into HEK293 cells, which express the CaSR, showed the mutant Ser220 Gα11 protein to impair CaSR-mediated Ca2+i and extracellular signal-regulated kinase 1/2 (ERK) MAPK signaling, consistent with diminished activation of PLC. Furthermore, engineered mutagenesis studies demonstrated that loss of hydrophobicity within the Gα11 cleft region also impaired signaling by PLC. The loss-of-function associated with the Ser220 Gα11 mutant was rectified by treatment of cells with cinacalcet, which is a CaSR-positive allosteric modulator. Furthermore, in vivo administration of cinacalcet to the proband harboring the Phe220Ser Gα11 mutation, normalized serum ionized calcium concentrations. Thus, our studies, which report a novel Gα11 germline mutation (Phe220Ser) in a family with FHH2, reveal the importance of the Gα11 hydrophobic cleft region for CaSR-mediated activation of PLC, and show that allosteric CaSR modulation can rectify the loss-of-function Phe220Ser mutation and ameliorate the hypercalcemia associated with FHH2. © 2017 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
Assuntos
Cinacalcete/uso terapêutico , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Mutação em Linhagem Germinativa/genética , Hipercalcemia/congênito , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Mutação com Perda de Função/genética , Adulto , Sequência de Aminoácidos , Cinacalcete/farmacologia , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipercalcemia/enzimologia , Hipercalcemia/patologia , Lactente , Recém-Nascido , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Linhagem , Fosforilação/efeitos dos fármacos , Transdução de Sinais , Fosfolipases Tipo C/metabolismoRESUMO
The gating of potassium ion (K+) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K+ channels, but also for developing the pharmaceutical compounds targeting K+ channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K+ channel and a G protein α subunit.