RESUMEN
Coordinating mitotic spindle dynamics with cortical polarity is essential for stem cell asymmetric divisions. Over the years, the protein Inscuteable (Insc) has emerged as a key element determining the spindle orientation in asymmetric mitoses. Its overexpression increases differentiative divisions in systems as diverse as mouse keratinocytes and radial glial cells. To date, the molecular explanation to account for this phenotype envisioned Insc as an adaptor molecule bridging between the polarity proteins Par3:Par6:aPKC and the spindle pulling machines assembled on NuMA:LGN:Gαi. However, recent biochemical and structural data revealed that Insc and NuMA are competitive interactors of LGN, challenging the simplistic idea of a single apical macromolecular complex, and demanding a revision of the actual working principles of Insc.
Asunto(s)
División Celular Asimétrica , Proteínas de Ciclo Celular/metabolismo , Animales , Pollos , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Queratinocitos/citología , Ratones , Neuroglía/citología , Fenotipo , Estructura Terciaria de Proteína , Ratas , Transducción de Señal , Huso Acromático/metabolismo , Células MadreRESUMEN
Coupling of spindle orientation to cellular polarity is a prerequisite for epithelial asymmetric cell divisions. The current view posits that the adaptor Inscuteable (Insc) bridges between Par3 and the spindle tethering machinery assembled on NuMALGNGαi(GDP), thus triggering apico-basal spindle orientation. The crystal structure of the Drosophila ortholog of LGN (known as Pins) in complex with Insc reveals a modular interface contributed by evolutionary conserved residues. The structure also identifies a positively charged patch of LGN binding to an invariant EPE-motif present on both Insc and NuMA. In vitro competition assays indicate that Insc competes with NuMA for LGN binding, displaying a higher affinity, and that it is capable of opening the LGN conformational switch. The finding that Insc and NuMA are mutually exclusive interactors of LGN challenges the established model of force generators assembly, which we revise on the basis of the newly discovered biochemical properties of the intervening components.
Asunto(s)
División Celular Asimétrica/fisiología , Proteínas del Citoesqueleto/genética , Proteínas de Drosophila/genética , Células Epiteliales/fisiología , Inhibidores de Disociación de Guanina Nucleótido/genética , Proteínas de la Membrana/genética , Modelos Moleculares , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Unión Competitiva/fisiología , Proteínas de Ciclo Celular , Polaridad Celular/fisiología , Pollos , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Cristalización , Proteínas del Citoesqueleto/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Electroforesis en Gel de Poliacrilamida , Polarización de Fluorescencia , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Oryzias , Conformación Proteica , Alineación de Secuencia , Huso Acromático/fisiología , Electricidad Estática , Xenopus laevis , Pez CebraRESUMEN
The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Cristalografía por Rayos X , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor/genéticaRESUMEN
Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.
RESUMEN
Inhibitor of growth protein 4 (ING4) belongs to the ING family of tumour suppressors and is involved in chromatin remodelling, in growth arrest and, in cooperation with p53, in senescence and apoptosis. Whereas the structure and histone H3-binding properties of the C-terminal PHD domains of the ING proteins are known, no structural information is available for the N-terminal domains. This domain contains a putative oligomerization site rich in helical structure in the ING2-5 members of the family. The N-terminal domain of ING4 was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments yielded crystals that were suitable for high-resolution X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 129.7, b = 188.3, c = 62.7 A. The self-rotation function and the Matthews coefficient suggested the presence of three protein dimers per asymmetric unit. The crystals diffracted to a resolution of 2.3 A using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).
Asunto(s)
Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Cristalización , Cristalografía por Rayos X , Humanos , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.
Asunto(s)
Antígenos Nucleares/metabolismo , Polaridad Celular , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Huso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/aislamiento & purificación , Células CACO-2 , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Microtúbulos/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/aislamiento & purificación , Unión Proteica/fisiología , Multimerización de Proteína/fisiología , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3-Insc-LGN-GαiGDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:GαiGDP suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , División Celular Asimétrica , Proteínas del Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/citología , Drosophila/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Madre/citología , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Drosophila/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Inhibidores de Disociación de Guanina Nucleótido/química , Inhibidores de Disociación de Guanina Nucleótido/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Unión Proteica , Células Madre/química , Células Madre/metabolismoRESUMEN
Streptococcus pneumoniae is an opportunistic respiratory pathogen that remains a major cause of morbidity and mortality globally, with infants and the elderly at the highest risk. S. pneumoniae relies entirely on carbohydrates as a source of carbon and dedicates a third of all uptake systems to carbohydrate import. The structure of the carbohydrate-free substrate-binding protein SP0092 at 1.61â Å resolution reveals it to belong to the newly proposed subclass G of substrate-binding proteins, with a ligand-binding pocket that is large enough to accommodate complex oligosaccharides. SP0092 is a dimer in solution and the crystal structure reveals a domain-swapped dimer with the monomer subunits in a closed conformation but in the absence of carbohydrate ligand. This closed conformation may be induced by dimer formation and could be used as a mechanism to regulate carbohydrate uptake.
Asunto(s)
Proteínas Bacterianas/química , Oligosacáridos/química , Receptores de Superficie Celular/química , Streptococcus pneumoniae/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Oligosacáridos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/metabolismo , Especificidad por SustratoRESUMEN
Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Receptores de Superficie Celular/química , Streptococcus pneumoniae/química , Proteínas Bacterianas/metabolismo , Estructura Molecular , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Streptococcus pneumoniae/metabolismoRESUMEN
Streptococcus pneumoniae is dependent on carbohydrate uptake for colonization and pathogenesis, and dedicates over a third of its transport systems to their uptake. The ability of the pneumococcus to utilize fructooligosaccharides (FOSs) is attributed to the presence of one of two types of FOS ATP-binding cassette (ABC) transporters. Strains encoding SfuABC are only able to utilize short-chain FOSs, while strains encoding FusABC can utilize both short- and long-chain FOSs. The crystal structures of the substrate-binding protein FusA in its open and closed conformations bound to FOSs, and solution scattering data of SfuA, delineate the structural basis for import of short- and long-chain FOSs. The structure of FusA identifies an EF hand-like calcium-binding motif. This is shown to be essential for translocation of FOSs in FusABC and forms the basis for the definition of a new class of substrate-binding proteins that regulate substrate translocation by calcium.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Calcio/metabolismo , Oligosacáridos/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Motivos EF Hand , Redes Reguladoras de Genes , Modelos Moleculares , Unión Proteica , Conformación Proteica , Streptococcus pneumoniae/química , Especificidad por SustratoRESUMEN
Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.