RESUMEN
Small-angle X-ray scattering (SAXS) has grown in popularity in recent times with the advent of bright synchrotron X-ray sources, powerful computational resources and algorithms enabling the calculation of increasingly complex models. However, the lack of standardized data-quality metrics presents difficulties for the growing user community in accurately assessing the quality of experimental SAXS data. Here, a series of metrics to quantitatively describe SAXS data in an objective manner using statistical evaluations are defined. These metrics are applied to identify the effects of radiation damage, concentration dependence and interparticle interactions on SAXS data from a set of 27 previously described targets for which high-resolution structures have been determined via X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The studies show that these metrics are sufficient to characterize SAXS data quality on a small sample set with statistical rigor and sensitivity similar to or better than manual analysis. The development of data-quality analysis strategies such as these initial efforts is needed to enable the accurate and unbiased assessment of SAXS data quality.
Asunto(s)
Dispersión del Ángulo Pequeño , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Proteínas/químicaRESUMEN
In this work we investigate the interplay between flow and boundary condition effects on the orientation field of a thermotropic nematic liquid crystal under flow and confinement in a microfluidic device. Two types of experiments were performed using synchrotron small-angle X-ray-scattering (SAXS). In the first, a nematic liquid crystal flows through a square-channel cross section at varying flow rates, while the nematic director orientation projected onto the velocity/velocity gradient plane is measured using a 2D detector. At moderate-to-high flow rates, the nematic director is predominantly aligned in the flow direction, but with a small tilt angle of â¼±11° in the velocity gradient direction. The director tilt angle is constant throughout most of the channel width but switches sign when crossing the center of the channel, in agreement with the Ericksen-Leslie-Parodi (ELP) theory. At low flow rates, boundary conditions begin to dominate, and a flow profile resembling the escaped radial director configuration is observed, where the director is seen to vary more smoothly from the edges (with homeotropic alignment) to the center of the channel. In the second experiment, hydrodynamic focusing is employed to confine the nematic phase into a sheet of liquid sandwiched between two layers of Triton X-100 aqueous solutions. The average nematic director orientation shifts to some extent from the flow direction toward the liquid boundaries, although it remains unclear if one tilt angle is dominant through most of the nematic sheet (with abrupt jumps near the boundaries) or if the tilt angle varies smoothly between two extreme values (â¼90 and 0°). The technique presented here could be applied to perform high-throughput measurements for assessing the influence of different surfactants on the orientation of nematic phases and may lead to further improvements in areas such as boundary lubrication and clarifying the nature of defect structures in LC displays.
Asunto(s)
Hidrodinámica , Dispositivos Laboratorio en un Chip , Cristales Líquidos/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Propiedades de SuperficieRESUMEN
Botulinum neurotoxins (BoNTs) are among the most poisonous biological substances known. They assemble with non-toxic non-hemagglutinin (NTNHA) protein to form the minimally functional progenitor toxin complexes (M-PTC), which protects BoNT in the gastrointestinal tract and releases it upon entry into the circulation. Here we provide molecular insight into the assembly between BoNT/A and NTNHA-A using small-angle X-ray scattering. We found that the free form BoNT/A maintains a pH-independent conformation with limited domain flexibility. Intriguingly, the free form NTNHA-A adopts pH-dependent conformational changes due to a torsional motion of its C-terminal domain. Once forming a complex at acidic pH, they each adopt a stable conformation that is similar to that observed in the crystal structure of the M-PTC. Our results suggest that assembly of the M-PTC depends on the environmental pH and that the complex form of BoNT/A is induced by interacting with NTNHA-A at acidic pH.
Asunto(s)
Toxinas Botulínicas/química , Neurotoxinas/química , Toxinas Botulínicas/metabolismo , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Neurotoxinas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
To evade host immune mechanisms, many bacteria secrete immunomodulatory enzymes. Streptococcus pyogenes, one of the most common human pathogens, secretes a large endoglycosidase, EndoS, which removes carbohydrates in a highly specific manner from IgG antibodies. This modification renders antibodies incapable of eliciting host effector functions through either complement or Fc γ receptors, providing the bacteria with a survival advantage. On account of this antibody-specific modifying activity, EndoS is being developed as a promising injectable therapeutic for autoimmune diseases that rely on autoantibodies. Additionally, EndoS is a key enzyme used in the chemoenzymatic synthesis of homogenously glycosylated antibodies with tailored Fc γ receptor-mediated effector functions. Despite the tremendous utility of this enzyme, the molecular basis of EndoS specificity for, and processing of, IgG antibodies has remained poorly understood. Here, we report the X-ray crystal structure of EndoS and provide a model of its encounter complex with its substrate, the IgG1 Fc domain. We show that EndoS is composed of five distinct protein domains, including glycosidase, leucine-rich repeat, hybrid Ig, carbohydrate binding module, and three-helix bundle domains, arranged in a distinctive V-shaped conformation. Our data suggest that the substrate enters the concave interior of the enzyme structure, is held in place by the carbohydrate binding module, and that concerted conformational changes in both enzyme and substrate are required for subsequent antibody deglycosylation. The EndoS structure presented here provides a framework from which novel endoglycosidases could be engineered for additional clinical and biotechnological applications.
Asunto(s)
Proteínas Bacterianas/química , Glicósido Hidrolasas/química , Inmunoglobulina G/metabolismo , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Glicósido Hidrolasas/inmunología , Glicósido Hidrolasas/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/química , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Streptococcus pyogenes/patogenicidad , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Dominio Catalítico , Proteínas Cromosómicas no Histona/química , Dimerización , Modelos Moleculares , Conformación Proteica , Proteína-Arginina N-Metiltransferasas/química , Especificidad por Sustrato , Proteínas de Xenopus/química , Xenopus laevisRESUMEN
The removal of chemically damaged DNA bases such as 3-methyladenine (3-MeA) is an essential process in all living organisms and is catalyzed by the enzyme 3-MeA DNA glycosylase I. A key question is how the enzyme selectively recognizes the alkylated 3-MeA over the much more abundant adenine. The crystal structures of native and Y16F-mutant 3-MeA DNA glycosylase I from Staphylococcus aureus in complex with 3-MeA are reported to 1.8 and 2.2 Å resolution, respectively. Isothermal titration calorimetry shows that protonation of 3-MeA decreases its binding affinity, confirming previous fluorescence studies that show that charge-charge recognition is not critical for the selection of 3-MeA over adenine. It is hypothesized that the hydrogen-bonding pattern of Glu38 and Tyr16 of 3-MeA DNA glycosylase I with a particular tautomer unique to 3-MeA contributes to recognition and selection.
Asunto(s)
Adenina/análogos & derivados , ADN Glicosilasas/química , Staphylococcus aureus/enzimología , Adenina/química , Adenina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Glicosilasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Especificidad por SustratoRESUMEN
The Technology Portal of the Protein Structure Initiative Structural Biology Knowledgebase (PSI SBKB; http://technology.sbkb.org/portal/ ) is a web resource providing information about methods and tools that can be used to relieve bottlenecks in many areas of protein production and structural biology research. Several useful features are available on the web site, including multiple ways to search the database of over 250 technological advances, a link to videos of methods on YouTube, and access to a technology forum where scientists can connect, ask questions, get news, and develop collaborations. The Technology Portal is a component of the PSI SBKB ( http://sbkb.org ), which presents integrated genomic, structural, and functional information for all protein sequence targets selected by the Protein Structure Initiative. Created in collaboration with the Nature Publishing Group, the SBKB offers an array of resources for structural biologists, such as a research library, editorials about new research advances, a featured biological system each month, and a functional sleuth for searching protein structures of unknown function. An overview of the various features and examples of user searches highlight the information, tools, and avenues for scientific interaction available through the Technology Portal.
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Biología Computacional/métodos , Bases de Datos de Proteínas , Internet , Sistemas en Línea , Conformación Proteica , Interfaz Usuario-Computador , Biología Computacional/instrumentación , Sistemas de Administración de Bases de Datos , Humanos , Bases del Conocimiento , Proteínas/química , Red SocialRESUMEN
Helicobacter pylori NikR (HpNikR) is a nickel-dependent transcription factor that regulates multiple genes in the H. pylori pathogen. There are conflicting data regarding the locations of the Ni(II) sites and the role of Ni(II) coordination in DNA recognition. Herein, we report crystal structures of (i) the metal-binding domain (MBD) of HpNikR (3.08 Å) and (ii) a mutant, H74A (2.04 Å), designed to disrupt native Ni(II) coordination. In the MBD structure, four nickel ions are coordinated to two different types of nickel sites (4-coordinate, square planar, and 5/6-coordinate, square pyramidal/octahedral). In the H74A structure, all four nickel ions are coordinated to 4-coordinate square-planar sites. DNA-binding studies reveal tighter binding for target DNA sequences for holo-HpNikR compared with the affinities of Ni(II) reconstituted apo-HpNikR and H74A for these same DNA targets, supporting a role for Ni(II) coordination to 5/6 sites in DNA recognition. Small-angle X-ray scattering studies of holo-HpNikR and H74A reveal a high degree of conformational flexibility centered at the DNA-binding domains of H74A, which is consistent with disorder observed in the crystal structure of the protein. A model of DNA recognition by HpNikR is proposed in which Ni(II) coordination to specific sites in the MBD have a long-range effect on the flexibility of the DNA-binding domains and, consequently, the DNA recognition properties.
Asunto(s)
Proteínas Bacterianas/metabolismo , Complejos de Coordinación/metabolismo , ADN/metabolismo , Helicobacter pylori/metabolismo , Níquel/metabolismo , Proteínas Represoras/metabolismo , Anisotropía , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X , Fluorescencia , Concentración de Iones de Hidrógeno , Modelos Biológicos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Estructura Terciaria de Proteína , Proteínas Represoras/química , Dispersión del Ángulo Pequeño , Terminología como Asunto , Difracción de Rayos XRESUMEN
The Protein Structure Initiative's Structural Biology Knowledgebase (SBKB, URL: http://sbkb.org ) is an open web resource designed to turn the products of the structural genomics and structural biology efforts into knowledge that can be used by the biological community to understand living systems and disease. Here we will present examples on how to use the SBKB to enable biological research. For example, a protein sequence or Protein Data Bank (PDB) structure ID search will provide a list of related protein structures in the PDB, associated biological descriptions (annotations), homology models, structural genomics protein target status, experimental protocols, and the ability to order available DNA clones from the PSI:Biology-Materials Repository. A text search will find publication and technology reports resulting from the PSI's high-throughput research efforts. Web tools that aid in research, including a system that accepts protein structure requests from the community, will also be described. Created in collaboration with the Nature Publishing Group, the Structural Biology Knowledgebase monthly update also provides a research library, editorials about new research advances, news, and an events calendar to present a broader view of structural genomics and structural biology.
Asunto(s)
Bases de Datos de Proteínas , Bases del Conocimiento , Sistemas en Línea , Proteínas/química , Secuencia de Aminoácidos , Sistemas de Administración de Bases de Datos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteómica , Interfaz Usuario-ComputadorRESUMEN
Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5'-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; D-cycloserine (DCS, also known as Seromycin) is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and L-cycloserine (LCS) is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5'-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5'-phosphate (PMP) and beta-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg(378) residue is involved in cycloserine inactivation of SPT.
Asunto(s)
Cicloserina/farmacología , Inhibidores Enzimáticos/farmacología , Serina C-Palmitoiltransferasa/antagonistas & inhibidores , Serina C-Palmitoiltransferasa/química , Serina C-Palmitoiltransferasa/metabolismo , Cromatografía Liquida , Cicloserina/química , Inhibidores Enzimáticos/química , Espectrometría de Masas , Estructura Molecular , Estructura Secundaria de Proteína , Serina C-Palmitoiltransferasa/genética , Sphingomonas/enzimologíaRESUMEN
The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology.
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Laboratorios/organización & administración , Proteínas/química , Proteínas/metabolismo , Proteómica/organización & administración , Biología Computacional , Cristalización , Humanos , Proteínas/genética , EscociaRESUMEN
The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha-beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.
Asunto(s)
Proteínas Bacterianas/química , Enzimas de Restricción-Modificación del ADN/antagonistas & inhibidores , Imitación Molecular , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , ADN/química , Desoxirribonucleasas de Localización Especificada Tipo I/antagonistas & inhibidores , Dimerización , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/químicaRESUMEN
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Recombinasas/antagonistas & inhibidores , Proteínas Arqueales/clasificación , Proteínas Arqueales/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/genética , Humanos , Isoenzimas/clasificación , Isoenzimas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Estructura Cuaternaria de Proteína , Recombinasas/metabolismo , Alineación de Secuencia , Sulfolobus solfataricus/enzimología , Sulfolobus solfataricus/genéticaRESUMEN
Sphingolipid biosynthesis begins with the condensation of L-serine and palmitoyl-CoA catalyzed by the PLP-dependent enzyme serine palmitoyltransferase (SPT). Mutations in human SPT cause hereditary sensory autonomic neuropathy type 1, a disease characterized by loss of feeling in extremities and severe pain. The human enzyme is a membrane-bound hetereodimer, and the most common mutations are located in the enzymatically incompetent monomer, suggesting a "dominant" or regulatory effect. The molecular basis of how these mutations perturb SPT activity is subtle and is not simply loss of activity. To further explore the structure and mechanism of SPT, we have studied the homodimeric bacterial enzyme from Sphingomonas paucimobilis. We have analyzed two mutants (N100Y and N100W) engineered to mimic the mutations seen in hereditary sensory autonomic neuropathy type 1 as well as a third mutant N100C designed to mimic the wild-type human SPT. The N100C mutant appears fully active, whereas both N100Y and N100W are significantly compromised. The structures of the holoenzymes reveal differences around the active site and in neighboring secondary structure that transmit across the dimeric interface in both N100Y and N100W. Comparison of the l-Ser external aldimine structures of both native and N100Y reveals significant differences that hinder the movement of a catalytically important Arg(378) residue into the active site. Spectroscopic analysis confirms that both N100Y and N100W mutants subtly affect the chemistry of the PLP. Furthermore, the N100Y and R378A mutants appear less able to stabilize a quinonoid intermediate. These data provide the first experimental insight into how the most common disease-associated mutations of human SPT may lead to perturbation of enzyme activity.
Asunto(s)
Serina C-Palmitoiltransferasa/química , Serina C-Palmitoiltransferasa/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Dominio Catalítico/genética , Cristalografía por Rayos X , Cartilla de ADN/genética , Neuropatías Hereditarias Sensoriales y Autónomas/enzimología , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Humanos , Iminas/química , Iminas/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Imitación Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina C-Palmitoiltransferasa/genética , Especificidad de la Especie , Espectrofotometría , Espectrofotometría Ultravioleta , Sphingomonas/enzimología , Sphingomonas/genética , Electricidad Estática , Especificidad por SustratoRESUMEN
Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ácido Cítrico/metabolismo , Dickeya chrysanthemi/metabolismo , Sideróforos/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Biocatálisis , Cromatografía Líquida de Alta Presión , Ácido Cítrico/química , Cartilla de ADN , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray , EstereoisomerismoRESUMEN
The Protein Structure Initiative Structural Genomics Knowledgebase (PSI SGKB, http://kb.psi-structuralgenomics.org) has been created to turn the products of the PSI structural genomics effort into knowledge that can be used by the biological research community to understand living systems and disease. This resource provides central access to structures in the Protein Data Bank (PDB), along with functional annotations, associated homology models, worldwide protein target tracking information, available protocols and the potential to obtain DNA materials for many of the targets. It also offers the ability to search all of the structural and methodological publications and the innovative technologies that were catalyzed by the PSI's high-throughput research efforts. In collaboration with the Nature Publishing Group, the PSI SGKB provides a research library, editorials about new research advances, news and an events calendar to present a broader view of structural biology and structural genomics. By making these resources freely available, the PSI SGKB serves as a bridge to connect the structural biology and the greater biomedical communities.
Asunto(s)
Bases de Datos de Proteínas , Conformación Proteica , Genómica , Proteínas/genética , Homología Estructural de ProteínaRESUMEN
Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 A resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.
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Sitio Alostérico , Bacillus anthracis/enzimología , Nucleósido-Fosfato Quinasa/química , Nucleótidos/metabolismo , Secuencia de Aminoácidos , Coenzimas/metabolismo , Cristalografía por Rayos X , Células Eucariotas/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Células Procariotas/enzimología , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Cathepsin L mutants with the ability to condense silica from solution have been generated and a 1.5 A crystal structure of one of these chimeras allows us to rationalise the catalytic mechanism of silicic acid condensation.
Asunto(s)
Catepsinas/química , Cisteína Endopeptidasas/química , Proteínas Recombinantes de Fusión/química , Catepsina L , Modelos Moleculares , Conformación ProteicaRESUMEN
TarO (http://www.compbio.dundee.ac.uk/taro) offers a single point of reference for key bioinformatics analyses relevant to selecting proteins or domains for study by structural biology techniques. The protein sequence is analysed by 17 algorithms and compared to 8 databases. TarO gathers putative homologues, including orthologues, and then obtains predictions of properties for these sequences including crystallisation propensity, protein disorder and post-translational modifications. Analyses are run on a high-performance computing cluster, the results integrated, stored in a database and accessed through a web-based user interface. Output is in tabulated format and in the form of an annotated multiple sequence alignment (MSA) that may be edited interactively in the program Jalview. TarO also simplifies the gathering of additional annotations via the Distributed Annotation System, both from the MSA in Jalview and through links to Dasty2. Routes to other information gateways are included, for example to relevant pages from UniProt, COG and the Conserved Domains Database. Open access to TarO is available from a guest account with private accounts for academic use available on request. Future development of TarO will include further analysis steps and integration with the Protein Information Management System (PIMS), a sister project in the BBSRC 'Structural Proteomics of Rational Targets' initiative.