RESUMEN
Chemotherapy-induced peripheral neuropathy (CIPN) is a major unmet medical need with limited treatment options. Despite different mechanisms of action, diverse chemotherapeutics can cause CIPN through a converged pathwayâan active axon degeneration program that engages the dual leucine zipper kinase (DLK). DLK is a neuronally enriched kinase upstream in the MAPK-JNK cascade, and while it is dormant under physiological conditions, DLK mediates a core mechanism for neuronal injury response under stress conditions, making it an attractive target for treatment of neuronal injury and neurodegenerative diseases. We have developed potent, selective, brain penetrant DLK inhibitors with excellent PK and activity in mouse models of CIPN. Lead compound IACS-52825 (22) showed strongly effective reversal of mechanical allodynia in a mouse model of CIPN and was advanced into preclinical development.
Asunto(s)
Antineoplásicos , Enfermedades del Sistema Nervioso Periférico , Ratones , Animales , Neuronas , Sistema de Señalización de MAP Quinasas , Encéfalo/metabolismo , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Antineoplásicos/efectos adversos , Quinasas Quinasa Quinasa PAMRESUMEN
cGMP-dependent protein kinase I-α (PKG1α) is a target for pulmonary arterial hypertension due to its role in the regulation of smooth muscle function. While most work has focused on regulation of cGMP turnover, we recently described several small molecule tool compounds which were capable of activating PKG1α via a cGMP independent pathway. Selected molecules were crystallized in the presence of PKG1α and were found to bind to an allosteric site proximal to the low-affinity nucleotide binding domain. These molecules act to displace the switch helix and cause activation of PKG1α representing a new mechanism for the activation and control of this critical therapeutic path. The described structures are vital to understanding the function and control of this key regulatory pathway.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismoRESUMEN
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Infecciones por VIH/tratamiento farmacológico , Antivirales/uso terapéutico , Apoptosis , Muerte Celular , Linfocitos T CD4-Positivos , Replicación ViralRESUMEN
Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I , GMP Cíclico , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Humanos , Miocitos del Músculo Liso , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.
Asunto(s)
Cristalización/métodos , Glicoproteínas de Membrana , Chaperonas Moleculares , Receptores Inmunológicos , Proteínas Recombinantes de Fusión , Humanos , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Receptores Inmunológicos/química , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3, ENPP3) is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. The full-length ectodomain of rat NPP3 was expressed in HEK293S GntI- cells, purified using two chromatographic steps and crystallized. Its structure at 2.77â Å resolution reveals that the active-site zinc ions are missing and a large part of the active site and the surrounding residues are flexible. The SMB-like domains have the same orientation in all four molecules in the asymmetric unit. The SMB2 domain is oriented as in NPP2, but the SMB1 domain does not interact with the PDE domain but extends further away from the PDE domain. Deletion of the SMB domains resulted in crystals that diffracted to 2.4â Å resolution and are suitable for substrate-binding studies.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Pirofosfatasas/química , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Células HEK293 , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Dominios Proteicos , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Somatomedinas/químicaRESUMEN
Ectonucleotide phosphodiesterase/pyrophosphatase-3 (NPP3) is a membrane-bound glycoprotein that regulates extracellular levels of nucleotides. NPP3 is known to contribute to the immune response on basophils by hydrolyzing ATP and to regulate the glycosyltransferase activity in Neuro2a cells. Here, we report on crystal structures of the nuclease and phosphodiesterase domains of rat NPP3 in complex with different substrates, products and substrate analogs giving insight into details of the catalytic mechanism. Complex structures with a phosphate ion, the product AMP and the substrate analog AMPNPP provide a consistent picture of the coordination of the substrate in which one zinc ion activates the threonine nucleophile whereas the other zinc ion binds the phosphate group. Co-crystal structures with the dinucleotide substrates Ap4A and UDPGlcNAc reveal a binding pocket for the larger leaving groups of these substrates. The crystal structures as well as mutational and kinetic analysis demonstrate that the larger leaving groups interact only weakly with the enzyme such that the substrate affinity is dominated by the interactions of the first nucleoside group. For this moiety, the nucleobase is stacked between Y290 and F207 and polar interactions with the protein are only formed via water molecules thus explaining the limited nucleobase selectivity.
RESUMEN
Kremen 1 and 2 have been identified as co-receptors for Dickkopf (Dkk) proteins, hallmark secreted antagonists of canonical Wnt signaling. We present here three crystal structures of the ectodomain of human Kremen1 (KRM1ECD) at resolutions between 1.9 and 3.2 Å. KRM1ECD emerges as a rigid molecule with tight interactions stabilizing a triangular arrangement of its Kringle, WSC, and CUB structural domains. The structures reveal an unpredicted homology of the WSC domain to hepatocyte growth factor. We further report the general architecture of the ternary complex formed by the Wnt co-receptor Lrp5/6, Dkk, and Krm, determined from a low-resolution complex crystal structure between ß-propeller/EGF repeats (PE) 3 and 4 of the Wnt co-receptor LRP6 (LRP6PE3PE4), the cysteine-rich domain 2 (CRD2) of DKK1, and KRM1ECD. DKK1CRD2 is sandwiched between LRP6PE3 and KRM1Kringle-WSC. Modeling studies supported by surface plasmon resonance suggest a direct interaction site between Krm1CUB and Lrp6PE2.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/química , Proteínas de la Membrana/química , Secuencias de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/ß-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8-mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling.
Asunto(s)
Proteínas del Ojo/química , Receptores Frizzled/química , Proteínas del Tejido Nervioso/química , Proteoglicanos/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Receptores Frizzled/metabolismo , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Conformación Proteica , Proteoglicanos/metabolismo , Dispersión del Ángulo PequeñoRESUMEN
The four secreted R-spondin (Rspo1-4) proteins of vertebrates function as stem cell growth factors and potentiate canonical Wnt signalling. Rspo proteins act by cross-linking members of two cell surface receptor families, complexing the stem cell markers LGR4-6 with the Frizzled-specific E3 ubiquitin ligases ZNRF3/RNF43. The consequent internalisation of the ternary LGR-Rspo-E3 complex removes the E3 ligase activity, which otherwise targets the Wnt receptor Frizzled for degradation, and thus enhances Wnt signalling. Multiple combinations of LGR4-6, Rspo1-4 and ZNRF3/RNF43 are possible, implying the existence of generic interaction determinants, but also of specific differences in complex architecture and activity. We present here a high resolution crystal structure of an ectodomain variant of human LGR5 (hLGR5ecto) complexed with a signalling competent fragment of mouse Rspo2 (mRspo2Fu1-Fu2). The structure shows that the particularly potent Rspo2 ligand engages LGR5 in a fashion almost identical to that reported for hRSPO1. Comparison of our hLGR5ecto structure with previously published structures highlights a surprising plasticity of the LGR ectodomains, characterised by a nearly 9° or larger rotation of the N-terminal half of the horseshoe-like fold relative to the C-terminal half. We also report a low resolution hLGR5-mRspo2Fu1-Fu2-mZNRF3ecto ternary complex structure. This crystal structure confirms our previously suggested hypothesis, showing that Rspo proteins cross-link LGRs and ZNRF3 into a 2:2:2 complex, whereas a 1:1:1 complex is formed with RNF43.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Trombospondinas/química , Ubiquitina-Proteína Ligasas/química , Sitios de Unión , Cristalografía por Rayos X , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
The interactions of extracellular ligands with single membrane spanning receptors, such as kinases, typically serve to agonise or antagonise the intracellular activation of signalling pathways. Within the cell, E3 ligases can act to alter the localisation and activity of proteins involved in signalling systems. Structural and functional characterisation of two closely related single membrane spanning molecules, RNF43 and ZNRF3, has recently revealed the receptor-like functionalities of a ligand-binding ectodomain combined with the intracellular architecture and activity of an E3 ligase. This direct link provides a hereto novel mechanism for extracellular control of ubiquitin ligase activity that is used for the modulation of Wnt signalling, a pathway of major importance in embryogenesis, stem cell biology and cancer. In this review we discuss recent findings for the structure and interactions of the extracellular region of RNF43/ZNRF3 and draw parallels with the properties and function of signalling receptor ectodomains.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Espacio Extracelular/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Unión al ADN/química , Humanos , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Terciaria de Proteína , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.
Asunto(s)
Carboxilesterasa/metabolismo , Proteínas de Drosophila/metabolismo , Esterasas/metabolismo , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Acilación , Animales , Sitios de Unión , Carboxilesterasa/química , Proteínas de Drosophila/química , Esterasas/química , Esterasas/genética , Ácidos Grasos Monoinsaturados/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Glipicanos/metabolismo , Humanos , Cinética , Ligandos , Espectrometría de Masas , Modelos Moleculares , Unión ProteicaRESUMEN
Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/ß hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.
Asunto(s)
Esterasas/genética , Cabeza/embriología , Neurogénesis/fisiología , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Ectodermo/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Silenciador del Gen , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Morfolinos , Placa Neural/metabolismo , Oxidación-Reducción , Ácido Palmítico/química , Unión Proteica , Conformación Proteica , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/química , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
The Arabidopsis E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase) AtAPY1 was previously shown to be involved in growth and development, pollen germination and stress responses. It was proposed to perform these functions through regulation of extracellular ATP signals. However, a GFP-tagged version was localized exclusively in the Golgi and did not hydrolyze ATP. In this study, AtAPY1 without the bulky GFP-tag was biochemically characterized with regard to its suggested role in purinergic signaling. Both the full-length protein and a soluble form without the transmembrane domain near the N-terminus were produced in HEK293 cells. Of the twelve nucleotide substrates tested, only three--GDP, IDP and UDP--were hydrolyzed, confirming that ATP was not a substrate of AtAPY1. In addition, the effects of pH, divalent metal ions, known E-NTPDase inhibitors and calmodulin on AtAPY1 activity were analyzed. AtAPY1-GFP extracted from transgenic Arabidopsis seedlings was included in the analyses. All three AtAPY1 versions exhibited very similar biochemical properties. Activity was detectable in a broad pH range, and Ca(2+), Mg(2+) and Mn(2+) were the three most efficient cofactors. Of the inhibitors tested, vanadate was the most potent one. Surprisingly, sulfonamide-based inhibitors shown to inhibit other E-NTPDases and presumed to inhibit AtAPY1 as well were not effective. Calmodulin stimulated the activity of the GFP-tagless membranous and soluble AtAPY1 forms about five-fold, but did not alter their substrate specificities. The apparent Km values obtained with AtAPY1-GFP indicate that AtAPY1 is primarily a GDPase. A putative three-dimensional structural model of the ecto-domain is presented, explaining the potent inhibitory potential of vanadate and predicting the binding mode of GDP. The found substrate specificity classifies AtAPY1 as a nucleoside diphosphatase typical of N-terminally anchored Golgi E-NTPDases and negates a direct function in purinergic signaling.
Asunto(s)
Apirasa/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Purinas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Apirasa/antagonistas & inhibidores , Apirasa/química , Apirasa/genética , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inhibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Calmodulina/metabolismo , Activación Enzimática , Expresión Génica , Guanosina Difosfato/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Nucleoside triphosphate diphosphohydrolases (NTPDases) are secreted or membrane-bound ectonucleotidases that hydrolyze the anhydride bonds of nucleoside triphosphates and nucleoside diphosphates. Mammalian cell-surface NTPDase enzymes are inhibited by various polyoxometallates. Here, the structures of NTPDase1 from the bacterium Legionella pneumophila (LpNTPDase1) in complex with the dodecatungstate POM-1, decavanadate and octamolybdate/heptamolybdate are described. The metal clusters are bound at different sites but always in a highly ordered fashion via electrostatic interactions and hydrogen bonds. For octamolybdate, covalent interactions after oxygen ligand exchange by a serine and histidine side chain are also observed. The potential inhibitory mechanism and the use of the metal clusters as phasing tools for new NTPDase structures are discussed. The binding mode of a tartrate ion at the catalytic centre suggests novel strategies for the structure-based design of NTPDase inhibitors, and the observation of the enzyme in an intermediate open state contributes to our understanding of NTPDase enzyme dynamics.
Asunto(s)
Antígenos CD/química , Apirasa/química , Legionella pneumophila/enzimología , Compuestos de Tungsteno/química , Antígenos CD/metabolismo , Apirasa/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Estructura Terciaria de Proteína , Homología Estructural de Proteína , Compuestos de Tungsteno/metabolismoRESUMEN
In many vertebrate tissues CD39-like ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) act in concert with ecto-5'-nucleotidase (e5NT, CD73) to convert extracellular ATP to adenosine. Extracellular ATP is a cytotoxic, pro-inflammatory signalling molecule whereas its product adenosine constitutes a universal and potent immune suppressor. Interference with these ectonucleotidases by use of small molecule inhibitors or inhibitory antibodies appears to be an effective strategy to enhance anti-tumour immunity and suppress neoangiogenesis. Here we present the first crystal structures of an NTPDase catalytic ectodomain in complex with the Reactive Blue 2 (RB2)-derived inhibitor PSB-071. In both of the two crystal forms presented the inhibitor binds as a sandwich of two molecules at the nucleoside binding site. One of the molecules is well defined in its orientation. Specific hydrogen bonds are formed between the sulfonyl group and the nucleoside binding loop. The methylphenyl side chain functionality that improved NTPDase2-specificity is sandwiched between R245 and R394, the latter of which is exclusively found in NTPDase2. The second molecule exhibits great in-plane rotational freedom and could not be modelled in a specific orientation. In addition to this structural insight into NTPDase inhibition, the observation of the putative membrane interaction loop (MIL) in two different conformations related by a 10° rotation identifies the MIL as a dynamic section of NTPDases that is potentially involved in regulation of catalysis.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Antraquinonas/química , Triazinas/químicaRESUMEN
The four R-spondin (Rspo) proteins are secreted agonists of Wnt signalling in vertebrates, functioning in embryogenesis and adult stem cell biology. Through ubiquitination and degradation of Wnt receptors, the transmembrane E3 ubiquitin ligase ZNRF3 and related RNF43 antagonize Wnt signalling. Rspo ligands have been reported to inhibit the ligase activity through direct interaction with ZNRF3 and RNF43. Here we report multiple crystal structures of the ZNRF3 ectodomain (ZNRF3(ecto)), a signalling-competent Furin1-Furin2 (Fu1-Fu2) fragment of Rspo2 (Rspo2(Fu1-Fu2)), and Rspo2(Fu1-Fu2) in complex with ZNRF3(ecto), or RNF43(ecto). A prominent loop in Fu1 clamps into equivalent grooves in the ZNRF3(ecto) and RNF43(ecto) surface. Rspo binding enhances dimerization of ZNRF3(ecto) but not of RNF43(ecto). Comparison of the four Rspo proteins, mutants and chimeras in biophysical and cellular assays shows that their signalling potency depends on their ability to recruit ZNRF3 or RNF43 via Fu1 into a complex with LGR receptors, which interact with Rspo via Fu2.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Oncogénicas/antagonistas & inhibidores , Trombospondinas/química , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Vía de Señalización Wnt , Animales , Proteínas de Unión al ADN/metabolismo , Dimerización , Humanos , Ratones , Proteínas Oncogénicas/metabolismo , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Xenopus , Pez CebraRESUMEN
Two nucleoside triphosphate diphosphohydrolase isoforms (NTPDase1 and NTPDase3) are responsible for the hydrolysis of nucleotides by the intracellular protozoan Toxoplasma gondii. They constitute about 3 % of the total parasite protein. Despite sharing 97 % sequence identity they exhibit opposite ATP versus ADP substrate discrimination ratios. Here we show by mutagenesis that the residues G492/G493 in NTPDase3 and R492/E493 in NTPDase1 are predominantly responsible for the differences in substrate specificity. Crystal structures of NTPDase1 in complexation with analogues of ATP and ADP reveal that the inverted substrate specificity of NTPDase1 relative to NTPDase3 is achieved by switching from the canonical substrate binding mode to a very different alternative one. Instead of being stacked on top of a helix of the C-terminal domain the nucleotide base is positioned in the interdomain space between the side chains of R108 and R492, recruited from both domains. Furthermore, we show that the NTPDase1 substrate specificity is mainly dependent on the presence of the side chain of E493, which causes repositioning of the ribose component of the nucleotide. All in all, binding by the flexible side chains in the alternative binding mode in NTPDase1 allows for equally good positioning of ATP and ADP with increased activity toward ADP relative to what is seen in the case of NTPDase3.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Pirofosfatasas/metabolismo , Toxoplasma/enzimología , Adenosina Difosfato/química , Adenosina Trifosfato/química , Antígenos CD/química , Antígenos CD/genética , Apirasa/química , Apirasa/genética , Sitios de Unión , Modelos Moleculares , Pirofosfatasas/química , Pirofosfatasas/genética , Especificidad por SustratoRESUMEN
In vertebrates, membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) on the cell surface are responsible for signal conversion and termination in purinergic signaling by extracellular nucleotides. Here we present apo and complex structures of the rat NTPDase2 extracellular domain and Legionella pneumophila NTPDase1, including a high-resolution structure with a transition-state analog. Comparison of ATP and ADP binding modes shows how NTPDases engage the same catalytic site for hydrolysis of nucleoside triphosphates and diphosphates. We find that this dual specificity is achieved at the expense of base specificity. Structural and mutational studies indicate that a conserved active-site water is replaced by the phosphate product immediately after phosphoryl transfer. Partial base specificity for purines in LpNTPDase1 is based on a different intersubunit base binding site for pyrimidine bases. A comparison of the bacterial enzyme in six independent crystal forms shows that NTPDases can undergo a domain closure motion of at least 17°.
Asunto(s)
Adenosina Trifosfatasas/química , Antígenos CD/química , Apirasa/química , Proteínas Bacterianas/química , Legionella pneumophila/enzimología , Secuencia de Aminoácidos , Animales , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Enlace de Hidrógeno , Cinética , Magnesio/química , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/química , Unión Proteica , Estructura Secundaria de Proteína , Ratas , Especificidad por SustratoRESUMEN
Nucleoside triphosphate diphosphohydrolases (NTPDases) are a large class of nucleotidases that hydrolyze the (γ/ß)- and (ß/α)-anhydride bonds of nucleoside triphosphates and diphosphates, respectively. NTPDases are found throughout the eukaryotic domain. In addition, a very small number of members can be found in bacteria, most of which live as parasites of eukaryotic hosts. NTPDases of intracellular and extracellular parasites are emerging as important regulators for the survival of the parasite. To deepen the knowledge of the structure and function of this enzyme class, recombinant production of the NTPDase1 from the bacterium Legionella pneumophila has been established. The protein could be crystallized in six crystal forms, of which one has been described previously. The crystals diffracted to resolutions of between 1.4 and 2.5â Å. Experimental phases determined by a sulfur SAD experiment using an orthorhombic crystal form produced an interpretable electron-density map.