RESUMO
Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.
Assuntos
Receptores Acoplados a Proteínas G , Transdução de Sinais , Humanos , Masculino , Microscopia Crioeletrônica , Sulfato de Desidroepiandrosterona , Desoxicorticosterona , Ligantes , Receptores Acoplados a Proteínas G/químicaRESUMO
The α2A adrenergic receptor (α2AAR) is a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that mediates important physiological functions in response to the endogenous neurotransmitters norepinephrine and epinephrine, as well as numerous chemically distinct drugs. However, the molecular mechanisms of drug actions remain poorly understood. Here, we report the cryo-electron microscopy structures of the human α2AAR-GoA complex bound to norepinephrine and three imidazoline derivatives (brimonidine, dexmedetomidine, and oxymetazoline). Together with mutagenesis and functional data, these structures provide important insights into the molecular basis of ligand recognition, activation, and signaling at the α2AAR. Further structural analyses uncover different molecular determinants between α2AAR and ßARs for recognition of norepinephrine and key regions that determine the G protein coupling selectivity. Overall, our studies provide a framework for understanding the signal transduction of the adrenergic system at the atomic level, which will facilitate rational structure-based discovery of safer and more effective medications for α2AAR.
Assuntos
Proteínas de Ligação ao GTP , Transdução de Sinais , Microscopia Crioeletrônica , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Norepinefrina , Transdução de Sinais/fisiologiaRESUMO
Many SARS-CoV-2 neutralizing antibodies (nAbs) lose potency against variants of concern. In this study, we developed 2 strategies to produce mutation-resistant antibodies. First, a yeast library expressing mutant receptor binding domains (RBDs) of the spike protein was utilized to screen for potent nAbs that are least susceptible to viral escape. Among the candidate antibodies, P5-22 displayed ultrahigh potency for virus neutralization as well as an outstanding mutation resistance profile. Additionally, P14-44 and P15-16 were recognized as mutation-resistant antibodies with broad betacoronavirus neutralization properties. P15-16 has only 1 binding hotspot, which is K378 in the RBD of SARS-CoV-2. The crystal structure of the P5-22, P14-44, and RBD ternary complex clarified the unique mechanisms that underlie the excellent mutation resistance profiles of these antibodies. Secondly, polymeric IgG enhanced antibody avidity by eliminating P5-22's only hotspot, residue F486 in the RBD, thereby potently blocking cell entry by mutant viruses. Structural and functional analyses of antibodies screened using both potency assays and the yeast RBD library revealed rare, ultrapotent, mutation-resistant nAbs against SARS-CoV-2.
Assuntos
Anticorpos Antivirais/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Afinidade de Anticorpos , Linfócitos B/imunologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Anticorpos Amplamente Neutralizantes/sangue , Anticorpos Amplamente Neutralizantes/genética , COVID-19/terapia , Clonagem Molecular , Modelos Animais de Doenças , Humanos , Imunização Passiva , Imunoglobulina G/imunologia , Técnicas In Vitro , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Testes de Neutralização , Receptores Virais/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Soroterapia para COVID-19RESUMO
Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.
Assuntos
Aciltransferases , Fallopia japonica/enzimologia , Proteínas de Plantas , Aciltransferases/biossíntese , Aciltransferases/química , Aciltransferases/genética , Aciltransferases/isolamento & purificação , Fallopia japonica/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoAssuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Bibliotecas de Moléculas Pequenas/farmacologia , Microscopia Crioeletrônica , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Homologia Estrutural de ProteínaRESUMO
The α2 adrenergic receptors (α2ARs) are G protein-coupled receptors (GPCRs) that respond to adrenaline and noradrenaline and couple to the Gi/o family of G proteins. α2ARs play important roles in regulating the sympathetic nervous system. Dexmedetomidine is a highly selective α2AR agonist used in post-operative patients as an anxiety-reducing, sedative medicine that decreases the requirement for opioids. As is typical for selective αAR agonists, dexmedetomidine consists of an imidazole ring and a substituted benzene moiety lacking polar groups, which is in contrast to ßAR-selective agonists, which share an ethanolamine group and an aromatic system with polar, hydrogen-bonding substituents. To better understand the structural basis for the selectivity and efficacy of adrenergic agonists, we determined the structure of the α2BAR in complex with dexmedetomidine and Go at a resolution of 2.9 Å by single-particle cryo-EM. The structure reveals the mechanism of α2AR-selective activation and provides insights into Gi/o coupling specificity.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 2/química , Dexmedetomidina/química , Receptores Adrenérgicos alfa 2/química , Receptores Adrenérgicos alfa 2/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Dexmedetomidina/metabolismo , Dexmedetomidina/farmacologia , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Insetos/citologia , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Receptores Adrenérgicos alfa 2/genética , Simpatolíticos/química , Simpatolíticos/farmacologiaRESUMO
Fusions to the C-terminal end of the Aga2p mating adhesion of Saccharomyces cerevisiae have been used in many studies for the selection of affinity reagents by yeast display followed by flow cytometric analysis. Here we present an improved yeast display system for the screening of Nanobody immune libraries where we fused the Nanobody to the N-terminal end of Aga2p to avoid steric hindrance between the fused Nanobody and the antigen. Moreover, the display level of a cloned Nanobody on the surface of an individual yeast cell can be monitored through a covalent fluorophore that is attached in a single enzymatic step to an orthogonal acyl carrier protein (ACP). Additionally, the displayed Nanobody can be easily released from the yeast surface and immobilised on solid surfaces for rapid analysis. To prove the generic nature of this novel Nanobody discovery platform, we conveniently selected Nanobodies against three different antigens, including two membrane proteins.
Assuntos
Moléculas de Adesão Celular , Biblioteca Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Anticorpos de Domínio Único , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Anticorpos de Domínio Único/biossíntese , Anticorpos de Domínio Único/genéticaRESUMO
The antibody IgE plays a central role in allergic disease, functioning principally through two cell-surface receptors: FcâRI and CD23. FcâRI on mast cells and basophils mediates the immediate hypersensitivity response, whilst the interaction of IgE with CD23 on B cells regulates IgE production. Crystal structures of the lectin-like `head' domain of CD23 alone and bound to a subfragment of IgE consisting of the dimer of Câ3 and Câ4 domains (Fcâ3-4) have recently been determined, revealing flexibility in the IgE-binding site of CD23. Here, a new crystal form of the CD23-Fcâ3-4 complex with different molecular-packing constraints is reported, which together with the earlier results demonstrates that conformational variability at the interface extends additionally to the IgE Fc and the quaternary structure of its domains.
Assuntos
Imunoglobulina E/química , Fragmentos Fc das Imunoglobulinas/química , Receptores de IgE/química , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcÉRI and CD23. Crosslinking of FcÉRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding.
Assuntos
Imunoglobulina E/química , Conformação Proteica , Receptores de Antígenos de Linfócitos B/química , Receptores de IgE/química , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sítios de Ligação/imunologia , Cristalografia por Raios X , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Modelos Moleculares , Ligação Proteica/imunologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de IgE/imunologia , Receptores de IgE/metabolismoRESUMO
Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcεRI and CD23 (FcεRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcεRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca(2+)-free and Ca(2+)-bound forms, as well as the crystal structure of the Ca(2+)-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). Together with site-directed mutagenesis, the crystal structures of four Ca(2+) ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca(2+) binds at the principal and evolutionarily conserved binding site in CD23. Ca(2+) binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca(2+)-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca(2+) brings an extra degree of modulation to CD23 function.
Assuntos
Linfócitos B/metabolismo , Cálcio/metabolismo , Imunoglobulina E/metabolismo , Receptores de IgE/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Ligação Competitiva , Calorimetria , Cristalografia por Raios X , Ciclofilina A/metabolismo , Entropia , Humanos , Imunoglobulina E/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Receptores de IgE/química , Receptores de IgE/genética , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
The role of IgE in allergic disease mechanisms is performed principally through its interactions with two receptors, FcεRI on mast cells and basophils, and CD23 (FcεRII) on B cells. The former mediates allergic hypersensitivity, the latter regulates IgE levels, and both receptors, also expressed on antigen-presenting cells, contribute to allergen uptake and presentation to the immune system. We have solved the crystal structure of the soluble lectin-like "head" domain of CD23 (derCD23) bound to a subfragment of IgE-Fc consisting of the dimer of Cε3 and Cε4 domains (Fcε3-4). One CD23 head binds to each heavy chain at the interface between the two domains, explaining the known 2:1 stoichiometry and suggesting mechanisms for cross-linking membrane-bound trimeric CD23 by IgE, or membrane IgE by soluble trimeric forms of CD23, both of which may contribute to the regulation of IgE synthesis by B cells. The two symmetrically located binding sites are distant from the single FcεRI binding site, which lies at the opposite ends of the Cε3 domains. Structural comparisons with both free IgE-Fc and its FcεRI complex reveal not only that the conformational changes in IgE-Fc required for CD23 binding are incompatible with FcεRI binding, but also that the converse is true. The two binding sites are allosterically linked. We demonstrate experimentally the reciprocal inhibition of CD23 and FcεRI binding in solution and suggest that the mutual exclusion of receptor binding allows IgE to function independently through its two receptors.
Assuntos
Imunoglobulina E/química , Complexos Multiproteicos/química , Receptores de IgE/química , Regulação Alostérica/imunologia , Linfócitos B/química , Linfócitos B/imunologia , Cristalografia por Raios X , Humanos , Imunoglobulina E/imunologia , Complexos Multiproteicos/imunologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de IgE/imunologia , Relação Estrutura-AtividadeRESUMO
Bifunctional pyrimidine deaminase/reductase (RibD) plays an important role during riboflavin biosynthesis in many microorganisms. The 40.4 kDa RibD from Shigella flexneri 2a has been cloned, expressed, purified and characterized. Three Crystals of RibD have been obtained by the hanging-drop technique at 291 K using PEG 20k or NaCl as precipitant. The RibD crystal using PEG 20k as precipitant diffracted to 2.5A.