RESUMEN
During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses. IMPORTANCE Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (49RKKRRQRRR57) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por VIH/virología , VIH-1/fisiología , Señales de Localización Nuclear , Dominios y Motivos de Interacción de Proteínas , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Secuencia de Consenso , Evolución Molecular , Interacciones Huésped-Patógeno , Modelos Moleculares , Unión Proteica , Transporte de Proteínas , Relación Estructura-Actividad , Proteínas Virales/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors.
RESUMEN
Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.
Asunto(s)
Cobre , Fotosíntesis/fisiología , Plastocianina , Populus , Cobre/química , Cobre/metabolismo , Citocromos f/química , Citocromos f/metabolismo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Complejo de Proteína del Fotosistema I/química , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Populus/química , Populus/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
It is generally assumed that the quality of X-ray diffraction data can be improved by merging data sets from several crystals. However, this effect is only valid if the data sets used are from crystals that are structurally identical. It is found that frozen macromolecular crystals very often have relatively low structure identity (and are therefore not isomorphous); thus, to obtain a real gain from multi-crystal data sets one needs to make an appropriate selection of structurally similar crystals. The application of hierarchical cluster analysis, based on the matrix of the correlation coefficient between scaled intensities, is proposed for the identification of isomorphous data sets. Multi-crystal single-wavelength anomalous dispersion data sets from four different protein molecules have been probed to test the applicability of this method. The use of hierarchical cluster analysis permitted the selection of batches of data sets which when merged together significantly improved the crystallographic indicators of the merged data and allowed solution of the structure.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/análisis , Animales , Bovinos , Análisis por Conglomerados , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
A reliable and reproducible method to automatically characterize the radiation sensitivity of macromolecular crystals at the ESRF beamlines has been developed. This new approach uses the slope of the linear dependence of the overall isotropic B-factor with absorbed dose as the damage metric. The method has been implemented through an automated procedure using the EDNA on-line data analysis framework and the MxCuBE data collection control interface. The outcome of the procedure can be directly used to design an optimal data collection strategy. The results of tests carried out on a number of model and real-life crystal systems are presented.
RESUMEN
To take into account the effects of radiation damage, new algorithms for the optimization of data-collection strategies have been implemented in the software package BEST. The intensity variation related to radiation damage is approximated by log-linear functions of resolution and cumulative X-ray dose. Based on an accurate prediction of the basic characteristics of data yet to be collected, BEST establishes objective relationships between the accessible data completeness, resolution and signal-to-noise statistics that can be achieved in an experiment and designs an optimal plan for data collection.
Asunto(s)
Cristalografía por Rayos X/métodos , Rayos X , Algoritmos , Aquifoliaceae/química , Hidrolasas de Éster Carboxílico/análisis , Clostridium thermocellum/enzimología , Relación Dosis-Respuesta en la Radiación , Insulina/análisis , Proteínas de la Membrana/análisis , ARN Interferente Pequeño/análisis , ARN Interferente Pequeño/química , Tombusvirus/química , Proteínas Virales/análisis , Proteínas Virales/químicaRESUMEN
The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.
Asunto(s)
Arthrobacter/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Cristalografía por Rayos X , Modelos Moleculares , Nicotina/análogos & derivados , Nicotina/metabolismo , Ácidos Fosfatidicos/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/químicaRESUMEN
EDNA is a framework for developing plugin-based applications especially for online data analysis in the X-ray experiments field. This article describes the features provided by the EDNA framework to ease the development of extensible scientific applications. This framework includes a plugins class hierarchy, configuration and application facilities, a mechanism to generate data classes and a testing framework. These utilities allow rapid development and integration in which robustness and quality play a fundamental role. A first prototype, designed for macromolecular crystallography experiments and tested at several synchrotrons, is presented.
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Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Programas Informáticos , Campylobacter jejuni/química , SincrotronesRESUMEN
Bacterial pentaheme cytochrome c nitrite reductases (NrfAs) are key enzymes involved in the terminal step of dissimilatory nitrite reduction of the nitrogen cycle. Their structure and functions are well studied. Recently, a novel octaheme cytochrome c nitrite reductase (TvNiR) has been isolated from the haloalkaliphilic bacterium Thioalkalivibrio nitratireducens. Here we present high-resolution crystal structures of the apoenzyme and its complexes with the substrate (nitrite) and the inhibitor (azide). Both in the crystalline state and in solution, TvNiR exists as a stable hexamer containing 48 hemes-the largest number of hemes accommodated within one protein molecule known to date. The subunit of TvNiR consists of two domains. The N-terminal domain has a unique fold and contains three hemes. The catalytic C-terminal domain hosts the remaining five hemes, their arrangement, including the catalytic heme, being identical to that found in NrfAs. The complete set of eight hemes forms a spatial pattern characteristic of other multiheme proteins, including structurally characterized octaheme cytochromes. The catalytic machinery of TvNiR resembles that of NrfAs. It comprises the lysine residue at the proximal position of the catalytic heme, the catalytic triad of tyrosine, histidine, and arginine at the distal side, channels for the substrate and product transport with a characteristic gradient of electrostatic potential, and, finally, two conserved Ca(2+)-binding sites. However, TvNiR has a number of special structural features, including a covalent bond between the catalytic tyrosine and the adjacent cysteine and the unusual topography of the product channels that open into the void interior space of the protein hexamer. The role of these characteristic structural features in the catalysis by this enzyme is discussed.
Asunto(s)
Proteínas Bacterianas/química , Citocromos a1/química , Citocromos c1/química , Ectothiorhodospiraceae/enzimología , Nitrato Reductasas/química , Estructura Cuaternaria de Proteína , Secuencia de Aminoácidos , Azidas/metabolismo , Cristalografía por Rayos X , Hemo/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nitritos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.
Asunto(s)
Glicoproteínas/metabolismo , Guanilato-Quinasas/química , Guanilato-Quinasas/metabolismo , Neuropéptidos/metabolismo , Animales , Línea Celular , Células Cultivadas , Cristalografía por Rayos X , Humanos , Magnesio/metabolismo , Ratones , Modelos Moleculares , Neuronas/metabolismo , Nucleótidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de AminoácidoRESUMEN
The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.
Asunto(s)
Adenosina Difosfato/química , Proteínas Bacterianas/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ácido Shikímico/análogos & derivados , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Unión Proteica , Estructura Terciaria de Proteína , Ácido Shikímico/química , Especificidad por SustratoRESUMEN
Statistical descriptors of the X-ray diffraction data set for a macromolecular crystal can be modelled using the information present in the initial diffraction images. Quantitative relationships between the crystal quality, beam apertures, oscillation width, resolution limit, redundancy and the data statistics are presented. They are analysed in terms of the radiation-dose requirements based on modelling in program the BEST. The influence of radiation damage on the data statistics is discussed.
Asunto(s)
Recolección de Datos , Gammaproteobacteria/enzimología , Modelos Estadísticos , Oxidorreductasas/química , Difracción de Rayos X/instrumentación , Aniones , Procesamiento Automatizado de Datos , Programas InformáticosRESUMEN
Triple-helical collagen IV protomers associate through their N- and C-termini, forming a three-dimensional network that provides basement membranes with mechanical strength. Within this network, the C-terminal non-collagenous (NC1) domains form tight dimeric junctions. Crystallographic analyses of isolated NC1 domains show two trimeric cap-like structures interacting via a large interface. Previously, for NC1 from human placenta type-IV collagen we described covalent alpha1-alpha1 and alpha2-alpha2 crosslinks between Met93 and Lys211 of opposing alpha1(IV) and alpha2(IV) NC1-chains, which further stabilize this interface and explain the occurrence of reduction-insensitive NC1-chain dimers. However, their existence was recently questioned, and we therefore analyzed NC1-domain dimers in more detail by biochemical and protein crystallographic methods. Short-exposure diffraction data show a clear electron density cross-connecting the respective residues, which gradually disappears with prolonged crystal irradiation. Sequence analyses of isolated tryptic peptides derived from denatured NC1 monomers and dimers indicate that only the dimers, but not the monomers, yield these chemically labile cross-linked peptides. These data clearly demonstrate the presence of reduction-resistant, but chemically and radiation-sensitive covalent crosslinks between the side chains of Met93 and Lys211 in human placenta type-IV collagen.
Asunto(s)
Membrana Basal/metabolismo , Colágeno Tipo IV/química , Reactivos de Enlaces Cruzados/química , Placenta/metabolismo , Secuencia de Aminoácidos , Dimerización , Femenino , Humanos , Lisina/química , Metionina/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Tripsina/metabolismoRESUMEN
Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.
Asunto(s)
Flavoproteínas/química , Secuencia de Aminoácidos , Arginina/química , Bacillus subtilis/metabolismo , Benzaldehídos/farmacología , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Dimerización , Electrones , Escherichia coli/metabolismo , Flavoproteínas/clasificación , Flavoproteínas/metabolismo , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Nitrofenoles/farmacología , Sistemas de Lectura Abierta , Estrés Oxidativo , Oxidorreductasas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tirosina/química , Difracción de Rayos XRESUMEN
The mammalian prohormone/proprotein convertase (PC) furin is responsible for the maturation of a great variety of homeostatic but also many pathogenic proteins within the secretory pathway and the endosomal pathway and at the cell surface. Similar to other members of the PC family, furin requires calcium for catalytic activity. In a previous paper, the structural association of the catalytic and the P-domain of furin was shown and data were presented indicating two or three calcium-binding sites. The exact number and the three-dimensional localization of the essential calcium sites within furin have now been determined by collecting X-ray diffraction data on either side of the Ca K absorption edge and by calculating a novel type of double difference map from these anomalous scattering data. Two calcium ions were unambiguously identified: the purely structural Ca-1 also conserved in the bacterial digestive subtilisins and the Ca-2 site specific to PCs and essential for the formation of the P1 specificity-determining S1-binding pocket. In addition, these anomalous diffraction data show that no tightly bound K(+) sites exist in furin.
Asunto(s)
Calcio/química , Furina/química , Cristalización , Cristalografía por Rayos X , Interpretación Estadística de Datos , Metales/química , Conformación Proteica , Especificidad por Sustrato , Subtilisinas/químicaRESUMEN
The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.
Asunto(s)
Bacteriófago lambda/metabolismo , Escherichia coli/metabolismo , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/metabolismo , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Termodinámica , Factores de Elongación TranscripcionalRESUMEN
Signaling pathways in eukaryotic cells are often controlled by the formation of specific signaling complexes, which are coordinated by scaffold and adaptor proteins. Elucidating their molecular architecture is essential to understand the spatial and temporal regulation of cellular signaling. p14 and MP1 form a tight (K(d) = 12.8 nM) endosomal adaptor/scaffold complex, which regulates mitogen-activated protein kinase (MAPK) signaling. Here, we present the 1.9-A crystal structure of a biologically functional p14/MP1 complex. The overall topology of the individual MP1 and p14 proteins is almost identical, having a central five-stranded beta-sheet sandwiched between a two-helix and a one-helix layer. Formation of the p14/MP1 heterodimer proceeds by beta-sheet augmentation and yields a unique, almost symmetrical, complex with several potential protein-binding sites on its surface. Mutational analysis allowed identification of the p14 endosomal adaptor motif, which seems to orient the complex relative to the endosomal membrane. Two highly conserved and hydrophobic protein-binding sites are located on the opposite "cytoplasmic" face of the p14/MP1 heterodimer and might therefore function as docking sites for the target proteins extracellular regulated kinase (ERK) 1 and MAPK/ERK kinase 1. Furthermore, detailed sequence analyses revealed that MP1/p14, together with profilins, define a protein superfamily of small subcellular adaptor proteins, named ProflAP. Taken together, the presented work provides insight into the spatial regulation of MAPK signaling, illustrating how p14 and MP1 collaborate as an endosomal adaptor/scaffold complex.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Endosomas/fisiología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas , Animales , Línea Celular , Clonación Molecular , Cricetinae , Cristalografía por Rayos X/métodos , Endosomas/enzimología , Células HeLa , Humanos , Ratones , Modelos Moleculares , Fosforilación , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
About one-third of the more than 300 C2H2 zinc finger proteins of Drosophila contain a conserved sequence motif, the zinc finger-associated domain (ZAD). Genes that encode ZAD proteins are specific for and expanded in the genomes of insects. Only three ZAD-encoding gene functions are established, and the role of ZAD is unknown. Here we present the crystal structure of the ZAD of Grauzone (ZAD(Grau)), a Drosophila transcription factor that specifically controls the maternal Cdc20-like APC subunit Cortex. ZAD forms an atypical treble-clef-like zinc-coordinating fold. Head-to-tail arrangement of two ZAD(Grau) molecules in the crystals suggests dimer formation, an observation supported by crosslinking and dynamic light scattering. The results indicate that ZAD provides a novel protein-protein interaction module that characterizes a large family of insect transcription factors.
Asunto(s)
Proteínas de Drosophila/química , Drosophila/metabolismo , Factores de Transcripción/química , Dedos de Zinc , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Anopheles , Dicroismo Circular , Reactivos de Enlaces Cruzados/farmacología , Cristalografía por Rayos X , ADN/química , Bases de Datos como Asunto , Dimerización , Drosophila melanogaster , Insectos , Luz , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción Genética , Zinc/químicaRESUMEN
Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas del Citoesqueleto/química , Dictyostelium/metabolismo , Secuencia de Aminoácidos , Animales , Dictyostelium/química , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
A new method and the software program BEST for optimal planning of X-ray data collection from protein crystals using the rotation method are presented. From one or a few initial diffraction images, BEST estimates the statistical characteristics of the data set for different combinations of data-collection parameters and suggests the most optimal ones. The anisotropy in diffraction and the permitted width of oscillation without spatially overlapping reflections are taken into account. According to the option chosen, the optimal set of parameters provides a given average signal-to-noise ratio at a given resolution either in the shortest time or with the minimum total radiation dose. BEST has been successfully used at the protein crystallography beamlines at DORIS (DESY). The software proved to be extremely useful in using the available data-collection time in the most efficient way.