RESUMEN
Lithium-ion batteries (LIBs) are among the most promising power sources for electric vehicles, portable electronics and smart grids. In LIBs, the cathode is a major bottleneck, with a particular reference to its low electrical conductivity and Li-ion diffusivity. The coating with carbon layers is generally employed to enhance the electrical conductivity and to protect the active material from degradation during operation. Here, we demonstrate that this layer has a primary role in the lithium diffusivity into the cathode nanoparticles. Positron is a useful quantum probe at the electroactive materials/carbon interface to sense the mobility of Li-ion. Broadband electrical spectroscopy demonstrates that only a small number of Li-ions are moving, and that their diffusion strongly depends on the type of carbon additive. Positron annihilation and broadband electrical spectroscopies are crucial complementary tools to investigate the electronic effect of the carbon phase on the cathode performance and Li-ion dynamics in electroactive materials.
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The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel ß-sheets organized into ß-sandwich layers. The amino-terminal domains 1-3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long ß-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4-6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/química , Insecticidas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escarabajos/efectos de los fármacos , Escarabajos/crecimiento & desarrollo , Cristalografía por Rayos X , Insecticidas/química , Intestinos/metabolismo , Larva/efectos de los fármacos , Larva/metabolismo , Mutagénesis Sitio-Dirigida , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Zea mays/metabolismo , Zea mays/parasitologíaRESUMEN
We tested the hypothesis that soil texture and nitrogen (N) fertilisation are the primary factors regulating the N cycle and soil bacterial community structure. The response of soil bacterial communities to N fertilisation in different textured soils might help in identifying the specific underlying mechanism and hence management of N fertiliser application in fields. We examined how N fertiliser accumulates in flue-cured tobacco and influences soil bacterial community structure in different textured soils. We conducted plot and micro-plot experimental measurements of N content in soil and tobacco samples using the KNO315N isotope technique. Soil bacterial community structure was determined using high-throughput sequencing of 16S rRNA. Nitrogen absorption and utilisation by tobacco plants were highest in sandy loam soils, followed by loam soil and clay loam. The ability of clay loam to supply N was weak during the plant growth period. Absence of fertilisation could reduce bacterial abundance in soils to various degrees. Bacterial diversity was higher in sandy loam soil than in loam soil and clay loam. Soil texture and N fertilisation significantly affected soil bacterial community structure and diversity. Proteobacteria, Acidobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Chloroflexi were the dominant bacterial phyla, while Bacillus, Nitrobacter, Nitrosospira, Nitrospira, and Rhizobium were the primary N transformation bacteria at the genus level in all treatments. However, relative abundances differed with N fertiliser application, which could lead to differential N availability and N use efficiency of tobacco among soil types. We conclude that both soil texture and N fertilisation influence N accumulation and distribution in flue-cured tobacco and thus regulate soil bacterial communities. N fertiliser application in sandy loam soil should be strictly controlled for its higher N use efficiency, soil bacterial abundance, and diversity.
RESUMEN
The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/farmacología , Insecticidas/farmacología , Control Biológico de Vectores/métodos , Plantas Modificadas Genéticamente , Zea mays , Animales , Proteínas Bacterianas/genética , Escarabajos/efectos de los fármacos , Resistencia a los Insecticidas/efectos de los fármacos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMEN
In recent years, NaFePO4 has been regarded as one of the most promising cathode materials for next-generation rechargeable sodium-ion batteries. There is significant interest in the redox processes of rechargeable batteries for high capacity applications. In this paper, the redox processes of triphylite-NaFePO4 and maricite-NaFePO4 materials have been analyzed based on first-principles calculations and analysis of Bader charges. Different from LiFePO4, anionic (O2-) redox reactions are evidently visible in NaFePO4. Electronic structures and density of states are calculated to elaborate the charge transfer and redox reactions during the desodiation processes. Furthermore, we also calculate the formation energies of sodium extraction, convex hull, average voltage plateaus, and volume changes of Na1-x/12FePO4 with different sodium compositions. Deformation charge density plots and magnetization for NaFePO4 are also calculated to help understand the redox reaction processes.
RESUMEN
Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full-length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening-over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate-binding domains, lectins, or from other insecticidal proteins.
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Bacillus thuringiensis/química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Pliegue de ProteínaRESUMEN
Temperature is a key element affecting the activity of microorganisms in activated sludge. Low water temperature generally leads to decreasing wastewater treatment efficiency and destroying sludge settleability. In this study, activated sludge samples from a municipal wastewater treatment plant (WWTP) implementing oxidation ditch process was used to investigate the bacterial community characteristics of a system that operates well in a cold region (Xinjiang, China) by high-throughput 16S rRNA gene sequencing. The results showed that the influent temperature was 7-12⯰C in winter and 13-17⯰C in summer, while the sludge volume index (SVI) of samples was between 51 and 74â¯mL/g. The average removal efficiencies for chemical oxygen demand (COD), biochemical oxygen demand (BOD5), suspended solid (SS), ammonia nitrogen (NH4+-N), total nitrogen (TN), and total phosphorus (TP) were 94%, 95%, 95%, 91%, 73% and 89%, respectively. The bacteria were distributed in 32 phyla and 559 genera. The dominant phyla were Proteobacteria (28.85-48.45%), Bacteroidetes (20.00-31.22%), Chloroflexi (3.59-12.23%), Actinobacteria (1.58-15.54%) and Firmicutes (1.38-10.49%). The dominant genera were Saprospiraceae_norank (4.41-12.23%), Comamonadaceae_unclassified (3.82-8.83%), Anaerolineaceae_norank (1.39-9.35%), Dokdonella (1.13-11.26%), Candidatus_Microthrix (0.26-7.50%), Flavobacterium (0.32-8.14%), Ferribacterium (0.36-5.19%) and Nitrospira (0.084-5.37%), which were different from those found in warm-region WWTPs. Contrary to previous studies, the relative abundance of ammonia-oxidizing bacteria (AOB; Nitrosomonas and Nitrosomonadaceae) and nitrite-oxidizing bacteria (NOB; Nitrospira) increased when the temperature decreased. The successful operation of this WWTP suggests that cold-region WWTPs can achieve good pollutants removal efficiency by simultaneously maintaining an ultra-low sludge load and high dissolved oxygen concentration in the oxidation ditch. The findings of this study provide fundamental knowledge required for an efficient and stable operation of WWTPs in cold regions.
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Bacterias , Microbiota , Eliminación de Residuos Líquidos/métodos , Oxidación-Reducción , Aguas del Alcantarillado/microbiología , TemperaturaRESUMEN
BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.
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Glycine max/enzimología , Resistencia a los Herbicidas/genética , Herbicidas/farmacología , Plantas Modificadas Genéticamente/enzimología , Zea mays/enzimología , Ingeniería Genética , Ácidos Indolacéticos/farmacología , Plantas Modificadas Genéticamente/genética , Propionatos/farmacología , Glycine max/genética , Zea mays/genéticaRESUMEN
The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.
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Proteínas Bacterianas/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Animales , Toxinas de Bacillus thuringiensis , Control de Insectos/métodosRESUMEN
LiCrO2 and NaCrO2 have been well-studied as cathode materials in lithium and sodium ion batteries, while the studies on LiCrS2 and NaCrS2 are relatively rare. In this work, a comparative study on the electronic structures and redox reactions in oxides (LiCrO2, NaCrO2) and sulfides (LiCrS2, NaCrS2) is performed. A first-principles method has been used to calculate the Bader charge transfer, the electronic structures and the magnetic moments during the entire delithiation or desodiation process. The Bader charge analysis suggests that all the S, O and Cr ions in LiCrX2 and NaCrX2 participate in the redox reactions, where the loss of electrons of S ions is clearly larger than that of O ions. Besides, the redox processes of Cr ions are of much less significance. It is noted that, in the sulfides, Cr ions even gain a small portion of electrons rather than losing electrons during the extraction of Li/Na ions. All the charge transfer happens between the S-3p/O-2p and the Cr-3d bands. The redox reactions of O or S ions originate from the energy levels of O/S being pushed towards/across the Fermi surface due to the strong p-d hybridization.
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The morphological structure and physiological indexes of Saccharomyces cerevisiae have changed during serial re-pitching due to the stress conditions in serial handlings and the cells become aging. It is of great significance to study the physiological changes of S. cerevisiae during serial re-pitching to understand the anti-aging effect of S. cerevisiae. In this paper, the changes of the physiological indexes during re-pitching of yeast are summarized, and based on the analysis of the previous works further research directions are proposed.
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Microbiología Industrial , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Técnicas MicrobiológicasRESUMEN
For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.
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Proteínas Bacterianas/química , Endotoxinas/química , Proteínas Hemolisinas/química , Insecticidas/química , Toxinas de Bacillus thuringiensis , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)-based motor. Dynactin binds both dynein and MTs via its p150(Glued) subunit, but little is known about the 'pointed-end complex' that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin-dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo-like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore-MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left-handed ß-helix domain, with the phosphorylation site located within a disordered, C-terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.
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Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Bovinos , Proteínas de Ciclo Celular/genética , Línea Celular , Embrión de Pollo , Complejo Dinactina , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Proteínas Proto-Oncogénicas/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Quinasa Tipo Polo 1RESUMEN
Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Glycine max/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Unión Proteica , Estructura Terciaria de Proteína , Eliminación de Secuencia , Glycine max/química , Glycine max/genéticaRESUMEN
The NudC family consists of four conserved proteins with representatives in all eukaryotes. The archetypal nudC gene from Aspergillus nidulans is a member of the nud gene family that is involved in the maintenance of nuclear migration. This family also includes nudF, whose human orthologue, Lis1, codes for a protein essential for brain cortex development. Three paralogues of NudC are known in vertebrates: NudC, NudC-like (NudCL), and NudC-like 2 (NudCL2). The fourth distantly related member of the family, CML66, contains a NudC-like domain. The three principal NudC proteins have no catalytic activity but appear to play as yet poorly defined roles in proliferating and dividing cells. We present crystallographic and NMR studies of the human NudC protein and discuss the results in the context of structures recently deposited by structural genomics centers (i.e., NudCL and mouse NudCL2). All proteins share the same core CS domain characteristic of proteins acting either as cochaperones of Hsp90 or as independent small heat shock proteins. However, while NudC and NudCL dimerize via an N-terminally located coiled coil, the smaller NudCL2 lacks this motif and instead dimerizes as a result of unique domain swapping. We show that NudC and NudCL, but not NudCL2, inhibit the aggregation of several target proteins, consistent with an Hsp90-independent heat shock protein function. Importantly, and in contrast to several previous reports, none of the three proteins is able to form binary complexes with Lis1. The availability of structural information will be of help in further studies on the cellular functions of the NudC family.
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Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Chaperonas Moleculares/metabolismo , Secuencia de Aminoácidos , Proteínas Fúngicas/química , Modelos MolecularesRESUMEN
The DH-PH domain tandems of Dbl-homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho-family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH-PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH-PH tandem of RhoA-specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH-PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide-free RhoA exhibits elevated dynamics when in complex with DH-PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.
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Factores de Intercambio de Guanina Nucleótido/química , Complejos Multiproteicos/química , Dominios PDZ , Proteína de Unión al GTP rhoA/química , Humanos , Conformación Proteica , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
BACKGROUND: The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Galpha12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG). RESULTS: Here we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition. CONCLUSION: Our results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.
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Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/química , Modelos Químicos , Animales , Dominio Catalítico , Humanos , Ratones , Mutación , Células 3T3 NIH , Dominios PDZ , Factores de Intercambio de Guanina Nucleótido RhoRESUMEN
The GntR superfamily of dimeric transcription factors, with more than 6200 members encoded in bacterial genomes, are characterized by N-terminal winged-helix DNA-binding domains and diverse C-terminal regulatory domains which provide a basis for the classification of the constituent families. The largest of these families, FadR, contains nearly 3000 proteins with all-alpha-helical regulatory domains classified into two related Pfam families: FadR_C and FCD. Only two crystal structures of FadR-family members, those of Escherichia coli FadR protein and LldR from Corynebacterium glutamicum, have been described to date in the literature. Here, the crystal structure of TM0439, a GntR regulator with an FCD domain found in the Thermotoga maritima genome, is described. The FCD domain is similar to that of the LldR regulator and contains a buried metal-binding site. Using atomic absorption spectroscopy and Trp fluorescence, it is shown that the recombinant protein contains bound Ni(2+) ions but that it is able to bind Zn(2+) with K(d) < 70 nM. It is concluded that Zn(2+) is the likely physiological metal and that it may perform either structural or regulatory roles or both. Finally, the TM0439 structure is compared with two other FadR-family structures recently deposited by structural genomics consortia. The results call for a revision in the classification of the FadR family of transcription factors.
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Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Proteínas Represoras/química , Thermotoga maritima/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Familia de Multigenes , Níquel/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/clasificación , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Thermotoga maritima/genética , Zinc/metabolismoRESUMEN
Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.