RESUMEN
The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.
Asunto(s)
Endonucleasas , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Transcripción Reversa , Humanos , Microscopía por Crioelectrón , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , ARN/genética , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Cristalografía por Rayos X , ADN/biosíntesis , ADN/genética , Inmunidad Innata , Interferones/biosíntesisRESUMEN
The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas c-met , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Mutación , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Antineoplásicos/farmacologíaRESUMEN
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Asunto(s)
Infecciones por VIH , VIH-1 , Alquinos , Benzoxazinas , Proteínas Adaptadoras de Señalización CARD/metabolismo , Ciclopropanos , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , VIH-1/metabolismo , Humanos , Inflamasomas/metabolismo , Leucocitos Mononucleares , Proteínas de Neoplasias/metabolismoRESUMEN
Herein the discovery of potent IDO1 inhibitors with low predicted human dose is discussed. Metabolite identification (MetID) and structural data were used to strategically incorporate cyclopropane rings into this tetrahydronaphthyridine series of IDO1 inhibitors to improve their metabolic stability and potency. Enabling synthetic chemistry was developed to construct these unique fused cyclopropyl compounds, leading to inhibitors with improved pharmacokinetics and human whole blood potency and a predicted human oral dose as low as 9 mg once daily (QD).
RESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Libraries of DNA-Encoded small molecules created using combinatorial chemistry and synthetic oligonucleotides are being applied to drug discovery projects across the pharmaceutical industry. The majority of reported projects describe the discovery of reversible, i.e. non-covalent, target modulators. We synthesized multiple DNA-encoded chemical libraries terminated in electrophiles and then used them to discover covalent irreversible inhibitors and report the successful discovery of acrylamide- and epoxide-terminated Bruton's Tyrosine Kinase (BTK) inhibitors. We also demonstrate their selectivity, potency and covalent cysteine engagement using a range of techniques including X-ray crystallography, thermal transition shift assay, reporter displacement assay and intact protein complex mass spectrometry. The epoxide BTK inhibitors described here are the first ever reported to utilize this electrophile for this target.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , ADN/química , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Agammaglobulinemia Tirosina Quinasa/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Indoleamine-2,3-dioxygenase-1 (IDO1) has emerged as an attractive target for cancer immunotherapy. An automated ligand identification system screen afforded the tetrahydroquinoline class of novel IDO1 inhibitors. Potency and pharmacokinetic (PK) were key issues with this class of compounds. Structure-based drug design and strategic incorporation of polarity enabled the rapid improvement on potency, solubility, and oxidative metabolic stability. Metabolite identification studies revealed that amide hydrolysis in the D-pocket was the key clearance mechanism for this class. Strategic survey of amide isosteres revealed that carbamates and N-pyrimidines, which maintained exquisite potencies, mitigated the amide hydrolysis issue and led to an improved rat PK profile. The lead compound 28 is a potent IDO1 inhibitor, with clean off-target profiles and the potential for quaque die dosing in humans.
RESUMEN
Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.
Asunto(s)
Adenosina Trifosfato/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleasas/metabolismo , Adenosina Trifosfato/química , Sitio Alostérico/efectos de los fármacos , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Endorribonucleasas/química , Técnicas de Inactivación de Genes , Humanos , Ligandos , Modelos Moleculares , Fosforilación , Conformación Proteica , Pliegue de Proteína , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Ribonucleasas/química , Respuesta de Proteína DesplegadaRESUMEN
Indoleamine-2,3-dioxygenase 1 (IDO1) inhibition and its combination with immune checkpoint inhibitors like pembrolizumab have drawn considerable attention from both academia and the pharmaceutical industry. Here, we describe the discovery of a novel class of highly potent IDO1 heme-displacing inhibitors featuring a unique bicyclo[1.1.1]pentane motif. Compound 1, evolving from an ALIS (automated ligand identification system) hit, exhibited excellent potency but lacked the desired pharmacokinetic profile due to extensive amide hydrolysis of the benzamide moiety. Replacing the central phenyl ring in 1 with a bicyclo[1.1.1]pentane bioisostere effectively circumvented the amide hydrolysis issue, resulting in the discovery of compound 2 with a favorable overall profile such as excellent potency, selectivity, pharmacokinetics, and a low predicted human dose.
RESUMEN
The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/farmacocinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Piperazinas/farmacología , Piperazinas/farmacocinética , Piperidinas/farmacología , Trasplante Homólogo , Administración Oral , Animales , Disponibilidad Biológica , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/metabolismo , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/metabolismo , Inmunosupresores/farmacocinética , Inmunosupresores/farmacología , Ratones , Simulación del Acoplamiento Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Piperazinas/administración & dosificación , Piperazinas/metabolismo , Piperidinas/administración & dosificación , Piperidinas/metabolismo , Conformación ProteicaRESUMEN
The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.
Asunto(s)
Factor Activador de Células B/química , Factor Activador de Células B/fisiología , Receptor del Factor Activador de Células B/química , Linfocitos B/citología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Factor Activador de Células B/genética , Diferenciación Celular , Supervivencia Celular , Reactivos de Enlaces Cruzados/química , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/química , Linfopenia/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) modulate B cell function by forming homotrimers and heterotrimers. To determine the structure of a heterotrimer of BAFF and APRIL, these ligands were expressed as a single chain protein in HEK 293 cells, purified by affinity and size exclusion chromatographies, and crystallized. Crystals belonging to the orthorhombic crystal system with a space group of C2221 diffracted to 2.43 Å. Initial structural solution was obtained by the molecular replacement method, and the structure was further refined to an R factor of 0.179 and free R factor of 0.234. The atomic coordinates and structure factors have been deposited into the Protein Data Bank (accession code 4ZCH).
RESUMEN
The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.
Asunto(s)
Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Factor Activador de Células B/química , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/genética , Dimerización , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Unión Proteica , Transducción de Señal , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.
Asunto(s)
Anticuerpos Neutralizantes/química , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Conformación Proteica , Factores de Necrosis Tumoral/química , Cristalografía , Citocina TWEAK , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Unión Proteica , Factores de Necrosis Tumoral/metabolismoRESUMEN
Several novel anti-CD20 monoclonal antibodies are currently in development with the aim of improving the treatment of B cell malignancies. Mutagenesis and epitope mapping studies have revealed differences between the CD20 epitopes recognized by these antibodies. Recently, X-ray crystallography studies confirmed that the Type I CD20 antibody rituximab and the Type II CD20 antibody obinutuzumab (GA101) differ fundamentally in their interaction with CD20 despite recognizing a partially overlapping epitope on CD20. The Type I CD20 antibodies rituximab and ofatumumab are known to bind to different epitopes. The differences suggest that the biological properties of these antibodies are not solely determined by their core epitope sequences, but also depend on other factors, such as the elbow hinge angle, the orientation of the bound antibody and differential effects mediated by the Fc region of the antibody. Taken together, these factors may explain differences in the preclinical properties and clinical efficacy of anti-CD20 antibodies.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos CD20/metabolismo , Epítopos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/metabolismo , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antígenos CD20/química , Antígenos CD20/genética , Antígenos CD20/inmunología , Ensayos Clínicos como Asunto , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/terapia , Linfoma no Hodgkin/terapia , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , RituximabRESUMEN
Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/administración & dosificación , Digoxigenina/inmunología , Sistemas de Liberación de Medicamentos/métodos , Anticuerpos Biespecíficos/inmunología , Antígenos de Neoplasias/inmunología , Carbocianinas/administración & dosificación , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Proteínas Fluorescentes Verdes/administración & dosificación , Humanos , Métodos , Anticuerpos de Cadena ÚnicaRESUMEN
CD20 is a cell-surface marker of normal and malignant B cells. Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by both types. We also determined the first X-ray structure of a type II antibody by crystallizing the obinutuzumab (GA101) Fab fragment alone and in complex with a CD20 cyclopeptide. Despite recognizing an overlapping epitope, GA101 binds CD20 in a completely different orientation than type I antibodies. Moreover, the elbow angle of GA101 is almost 30° wider than in type I antibodies, potentially resulting in different spatial arrangements of 2 CD20 molecules bound to a single GA101 or rituximab molecule. Using protein tomography, different CD20 complexes were found to be associated with the 2 antibodies, and confocal microscopy showed different membrane compartmentalization of these subpopulations of the cellular CD20 pool. Our findings offer a possible molecular explanation for the different cellular responses elicited by type I and II antibodies.
Asunto(s)
Anticuerpos Monoclonales/clasificación , Antígenos CD20/química , Antígenos CD20/inmunología , Epítopos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino/química , Especificidad de Anticuerpos , Antígenos CD20/genética , Línea Celular , Cristalografía por Rayos X , Mapeo Epitopo/métodos , Epítopos/análisis , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , RituximabRESUMEN
ATP-binding cassette (ABC) enzymes are involved in diverse biological processes ranging from transmembrane transport to chromosome cohesion and DNA repair. They typically use ATP hydrolysis to conduct energy-dependent biological reactions. However, the cystic fibrosis transmembrane conductance regulator and the DNA repair protein Rad50 can also catalyze the adenylate kinase reaction (ATP+AMP<-->2ADP). To clarify and provide a mechanistic basis for the adenylate kinase activity of ABC enzymes, we report the crystal structure of the nucleotide-binding domain of the Pyrococcus furiosus structural maintenance of chromosome protein (pfSMC(nbd)) in complex with the adenylate kinase inhibitor P(1),P(5)-di(adenosine-5')pentaphosphate. We show that pfSMC(nbd) possesses reverse adenylate kinase activity. Our results suggest that in adenylate kinase reactions, ATP binds to its canonical binding site while AMP binds to the Q-loop glutamine and a hydration water of the Mg(2+) ion. Furthermore, mutational analysis indicates that adenylate kinase reaction occurs in the engaged pfSMC(nbd) dimer and requires the Signature motif for phosphate transfer. Our results explain how ATP hydrolysis and adenylate kinase reactions can be catalyzed by the same functional motifs within the structural framework of ABC enzymes. Thus, adenylate kinase activity is likely to be a latent activity in many ABC enzymes.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenilato Quinasa/química , Adenilato Quinasa/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Nucleótidos de Adenina/metabolismo , Adenosina Trifosfatasas/genética , Adenilato Quinasa/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Modelos Moleculares , Conformación Proteica , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismoRESUMEN
RIG-I and MDA5 sense cytoplasmic viral RNA and set-off a signal transduction cascade, leading to antiviral innate immune response. The third RIG-I-like receptor, LGP2, differentially regulates RIG-I- and MDA5-dependent RNA sensing in an unknown manner. All three receptors possess a C-terminal regulatory domain (RD), which in the case of RIG-I senses the viral pattern 5'-triphosphate RNA and activates ATP-dependent signaling by RIG-I. Here we report the 2.6 A crystal structure of LGP2 RD along with in vitro and in vivo functional analyses and a homology model of MDA5 RD. Although LGP2 RD is structurally related to RIG-I RD, we find it rather binds double-stranded RNA (dsRNA) and this binding is independent of 5'-triphosphates. We identify conserved and receptor-specific parts of the RNA binding site. Latter are required for specific dsRNA binding by LGP2 RD and could confer pattern selectivity between RIG-I-like receptors. Our data furthermore suggest that LGP2 RD modulates RIG-I-dependent signaling via competition for dsRNA, another pattern sensed by RIG-I, while a fully functional LGP2 is required to augment MDA5-dependent signaling.