RESUMO
We report the x-ray crystal structure of intact, full-length human immunoglobulin (IgG4) at 1.8 Å resolution. The data for IgG4 (S228P), an antibody targeting the natriuretic peptide receptor A, show a previously unrecognized type of Fab-Fc orientation with a distorted λ-shape in which one Fab-arm is oriented toward the Fc portion. Detailed structural analysis by x-ray crystallography and molecular simulations suggest that this is one of several conformations coexisting in a dynamic equilibrium state. These results were confirmed by small angle x-ray scattering in solution. Furthermore, electron microscopy supported these findings by preserving molecule classes of different conformations. This study fosters our understanding of IgG4 in particular and our appreciation of antibody flexibility in general. Moreover, we give insights into potential biological implications, specifically for the interaction of human anti-natriuretic peptide receptor A IgG4 with the neonatal Fc receptor, Fcγ receptors, and complement-activating C1q by considering conformational flexibility.
Assuntos
Anticorpos/química , Imunoglobulina G/química , Receptores do Fator Natriurético Atrial/imunologia , Animais , Sítios de Ligação , Células CHO , Cricetulus , Cristalização , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptores de IgG/químicaRESUMO
Brain-derived neurotrophic factor (BDNF) is a central modulator of neuronal development and synaptic plasticity in the central nervous system. This renders the BDNF-modulated tropomyosin receptor kinase B (TrkB) a promising drug target to treat synaptic dysfunctions. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the so-called GRINCH neurons were derived from human-induced pluripotent stem cells. These GRINCH neurons were used as model cells for pharmacologic profiling of two TrkB-agonistic antibodies, hereafter referred to as AB2 and AB20 In next-generation sequencing studies, AB2 and AB20 stimulated transcriptional changes, which extensively overlapped with BDNF-driven transcriptional modulation. In regard to TrkB phosphorylation, both AB2 and AB20 were only about half as efficacious as BDNF; however, with respect to the TrkB downstream signaling, AB2 and AB20 displayed increased efficacy values, providing a stimulation at least comparable to BDNF in respect to VGF transcription, as well as of AKT and cAMP response element-binding protein phosphorylation. In a complex structure of the TrkB-d5 domain with AB20, determined by X-ray crystallography, the AB20 binding site was found to be allosteric in regard to the BDNF binding site, whereas AB2 was known to act orthosterically with BDNF. In agreement with this finding, AB2 and AB20 acted synergistically at greater concentrations to drive TrkB phosphorylation. Although TrkB downstream signaling declined faster after pulse stimulation with AB20 than with AB2, AB20 restimulated TrkB phosphorylation more efficiently than AB2. In conclusion, both antibodies displayed some limitations and some benefits in regard to future applications as therapeutic agents.
Assuntos
Anticorpos Monoclonais/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptor trkB/agonistas , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Fator Neurotrófico Derivado do Encéfalo/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor trkB/química , Receptor trkB/metabolismoRESUMO
Keap1 is a substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex and plays an important role in the cellular response to oxidative stress. It binds Nrf2 with its Kelch domain and thus triggers the ubiquitinylation and degradation of Nrf2. Oxidative stress prevents the degradation of Nrf2 and leads to the activation of cytoprotective genes. Therefore, Keap1 is an attractive drug target in inflammatory diseases. The support of a medicinal chemistry effort by structural research requires a robust crystallization system in which the crystals are preferably suited for performing soaking experiments. This facilitates the generation of protein-ligand complexes in a routine and high-throughput manner. The structure of human Keap1 has been described previously. In this crystal form, however, the binding site for Nrf2 was blocked by a crystal contact. This interaction was analysed and mutations were introduced to disrupt this crystal contact. One double mutation (E540A/E542A) crystallized in a new crystal form in which the binding site for Nrf2 was not blocked and was accessible to small-molecule ligands. The crystal structures of the apo form of the mutated Keap1 Kelch domain (1.98 Å resolution) and of the complex with an Nrf2-derived peptide obtained by soaking (2.20 Å resolution) are reported.
Assuntos
Proteínas de Transporte/química , Engenharia Química/métodos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalização , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Ligantes , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genéticaRESUMO
Polyclonal autoantibodies against human GM-CSF (granulocyte/macrophage colony-stimulating factor) are a hallmark of PAP (pulmonary alveolar proteinosis) and several other reported autoimmune diseases. MB007 is a high-affinity anti-(human GM-CSF) autoantibody isolated from a patient suffering from PAP which shows only modest neutralization of GM-CSF bioactivity. We describe the first crystal structure of a cytokine-directed human IgG1λ autoantibody-binding fragment (Fab) at 1.9 Å (1 Å=0.1 nm) resolution. Its CDR3-H substantially differs from all VH7 germline IgG1 structures reported previously. We derive a reliable model of the antigen-autoantibody complex by using NMR chemical shift perturbation data in combination with computational methods. Superposition of the modelled complex structure with the human GM-CSF-GM-CSF ternary receptor complex reveals only little overlap between receptor and Fab when bound to GM-CSF. Our model provides a structural basis for understanding the mode of action of the MB007 autoantibody.
Assuntos
Autoanticorpos/química , Autoanticorpos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Imunoglobulina G/química , Proteinose Alveolar Pulmonar/imunologia , Complexo Antígeno-Anticorpo/química , Autoanticorpos/uso terapêutico , Sítios de Ligação de Anticorpos/efeitos dos fármacos , Cristalização , Mapeamento de Epitopos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Modelos MolecularesRESUMO
Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all alpha-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.
Assuntos
Proteínas de Bactérias/química , Proteínas Repressoras/química , Vibrio parahaemolyticus/química , Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Óperon/fisiologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/biossíntese , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Shigella flexneri/genética , Shigella flexneri/metabolismo , Homologia Estrutural de Proteína , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
A new angucyclinone antibiotic, frigocyclinone, was isolated from Streptomyces griseus strain NTK 97, consisting of a tetrangomycin moiety attached through a C-glycosidic linkage with the aminodeoxysugar ossamine. Frigocyclinone showed antibacterial activities against Gram-positive bacteria.
Assuntos
Antraquinonas/farmacologia , Antibacterianos/farmacologia , Streptomyces griseus/metabolismo , Regiões Antárticas , Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Fermentação , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Espectrofotometria UltravioletaRESUMO
AMP-activated kinase (AMPK) is central to sensing energy status in eukaryotic cells via binding of AMP and ATP to CBS (cystathionine beta-synthase) domains in the regulatory gamma subunit. The structure of a CBS-domain pair from human AMPK gamma1 in complex with the physiological activator AMP and the pharmacological activator ZMP (AICAR) is presented.