Effect of long-term mechanical perturbation on intertidal soft-bottom meiofaunal community spatial structure

NASA Astrophysics Data System (ADS)

Boldina, Inna; Beninger, Peter G.; Le Coz, Maïwen

2014-01-01

Situated at the interface of the microbial and macrofaunal compartments, soft-bottom meiofauna accomplish important ecological functions. However, little is known of their spatial distribution in the benthic environment. To assess the effects of long-term mechanical disturbance on soft-bottom meiofaunal spatial distribution, we compared a site subjected to long-term clam digging to a nearby site untouched by such activities, in Bourgneuf Bay, on the Atlantic coast of France. Six patterned replicate samples were taken at 3, 6, 9, 12, 15, 18, 21 and 24 cm lags, all sampling stations being separated by 5 m. A combined correlogram-variogram approach was used to enhance interpretation of the meiofaunal spatial distribution; in particular, the definition of autocorrelation strength and its statistical significance, as well as the detailed characteristics of the periodic spatial structure of nematode assemblages, and the determination of the maximum distance of their spatial autocorrelation. At both sites, nematodes and copepods clearly exhibited aggregated spatial structure at the meso scale; this structure was attenuated at the impacted site. The nematode spatial distribution showed periodicity at the non-impacted site, but not at the impacted site. This is the first explicit report of a periodic process in meiofaunal spatial distribution. No such cyclic spatial process was observed for the more motile copepods at either site. This first study to indicate the impacts of long-term anthropogenic mechanical perturbation on meiofaunal spatial structure opens the door to a new dimension of mudflat ecology. Since macrofaunal predator search behaviour is known to be strongly influenced by prey spatial structure, the alteration of this structure may have important consequences for ecosystem functioning.

Modulation of individual steps in group I intron catalysis by a peripheral metal ion.

PubMed

Forconi, Marcello; Piccirilli, Joseph A; Herschlag, Daniel

2007-10-01

Enzymes are complex macromolecules that catalyze chemical reactions at their active sites. Important information about catalytic interactions is commonly gathered by perturbation or mutation of active site residues that directly contact substrates. However, active sites are engaged in intricate networks of interactions within the overall structure of the macromolecule, and there is a growing body of evidence about the importance of peripheral interactions in the precise structural organization of the active site. Here, we use functional studies, in conjunction with published structural information, to determine the effect of perturbation of a peripheral metal ion binding site on catalysis in a well-characterized catalytic RNA, the Tetrahymena thermophila group I ribozyme. We perturbed the metal ion binding site by site-specifically introducing a phosphorothioate substitution in the ribozyme's backbone, replacing the native ligands (the pro-R (P) oxygen atoms at positions 307 and 308) with sulfur atoms. Our data reveal that these perturbations affect several reaction steps, including the chemical step, despite the absence of direct contacts of this metal ion with the atoms involved in the chemical transformation. As structural probing with hydroxyl radicals did not reveal significant change in the three-dimensional structure upon phosphorothioate substitution, the effects are likely transmitted through local, rather subtle conformational rearrangements. Addition of Cd(2+), a thiophilic metal ion, rescues some reaction steps but has deleterious effects on other steps. These results suggest that native interactions in the active site may have been aligned by the naturally occurring peripheral residues and interactions to optimize the overall catalytic cycle.

Abiotic Factors Affecting Benthic Invertebrate Biomass and Community Structure in a Fourth-Order Rocky Mountain Watershed

NASA Astrophysics Data System (ADS)

Chanat, J. G.; Clements, W. H.; MacDonald, L. H.

2005-05-01

The potential ecological impact of excess streambed sediment resulting from forest management activities is a persistent concern for land managers. This study examined the relationship between streambed sediment, along with other site- and reach-scale abiotic factors, and benthic macroinvertebrate community structure in a 272 km2 basin in the Colorado Front Range. Physical habitat parameters and invertebrates were sampled in late summer at 68 sites located in sixteen stream reaches. Invertebrate data were used to formulate twenty indices of community structure. Multiple regression identified site-level substrate particle size as the most important predictor of six indices, including total density (R2 = 0.22), biomass (R2 = 0.17), and taxa richness (R2 = 0.32). All of the remaining fourteen indices were most strongly predicted by reach-level variables, including discharge (percent shredders, R2 = 0.24; Plecoptera density, R2 = 0.29), and elevation (percent collector-filterers, R2 = 0.28; Trichoptera density, R2 = 0.37). Although the sites represented a wide range of substrate composition and embeddedness, no physical variable associated with fine sediment appeared as a strong predictor of any of the twenty indices. Thus, sediment is not among the most important factors associated with site-to-site variability of benthic community structure in this relatively pristine watershed.

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Soil conservation through sediment trapping: A review

NASA Astrophysics Data System (ADS)

Mekonnen, Mulatie; Keesstra, Saskia; Baartman, Jantiene; Maroulis, Jerry; Stroosnijder, Leo

2014-05-01

Preventing the off-site effects of soil erosion is an essential part of good catchment management. Most efforts are in the form of on-site soil and water conservation measures. However, sediment trapping (ST) can be an alternative (or additional) measure to prevent the negative off-site effects of soil erosion. Therefore, not all efforts should focus solely on on-site soil conservation, but also on the safe routing of sediment-laden flows and on creating sites and conditions where sediment can be trapped, preferably in a cost effective or even profitable way. ST can be applied on-site (in-field) and off-site and involves both vegetative and structural measures. The main vegetative measures include grass strips, tree or bush buffers, grassed waterways and restoration of the waterways and their riparian zone; while structural measures include terraces, ponds and check dams. This paper provides a review of studies that have assessed the sediment trapping efficacy (STE) of such vegetative and structural measures. Vegetation type and integration of two or more measures (vegetative as well as structural) are important factors influencing STE. In this review, the STE of most measures was evaluated either individually or in such combinations. In real landscape situations, it is not only important to select the most efficient erosion control measures, but also to determine their optimum location in the catchment. Hence, there is a need for research that shows a more integrated determination of STE at the catchment scale. If integrated measures are implemented at the most appropriate spatial locations within a catchment where they can disconnect landscape units from each other, they will decrease runoff velocity and sediment transport and, subsequently, reduce downstream flooding and sedimentation problems. KEY WORDS: Integrated sediment trapping, sediment trapping efficacy, vegetative, structural, on-site and off-site measures.

Discriminative structural approaches for enzyme active-site prediction.

PubMed

Kato, Tsuyoshi; Nagano, Nozomi

2011-02-15

Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

Normal Modes Expose Active Sites in Enzymes.

PubMed

Glantz-Gashai, Yitav; Meirson, Tomer; Samson, Abraham O

2016-12-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes.

Normal Modes Expose Active Sites in Enzymes

PubMed Central

Glantz-Gashai, Yitav; Samson, Abraham O.

2016-01-01

Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

PubMed

Krishnan, Neeraja M; Seligmann, Hervé; Rao, Basuthkar J

2008-01-28

Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence, full structure, and active site microenvironment similarity.

PubMed

Leuthaeuser, Janelle B; Knutson, Stacy T; Kumar, Kiran; Babbitt, Patricia C; Fetrow, Jacquelyn S

2015-09-01

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods. © 2015 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence, full structure, and active site microenvironment similarity

PubMed Central

Leuthaeuser, Janelle B; Knutson, Stacy T; Kumar, Kiran; Babbitt, Patricia C; Fetrow, Jacquelyn S

2015-01-01

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods. PMID:26073648

Library Web Site Administration: A Strategic Planning Model For the Smaller Academic Library

ERIC Educational Resources Information Center

Ryan, Susan M.

2003-01-01

Strategic planning provides a useful structure for creating and implementing library web sites. The planned integration of a library's web site into its mission and objectives ensures that the library's community of users will consider the web site one of the most important information tools the library offers.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Territory and nest site selection patterns by Grasshopper Sparrows in southeastern Arizona

USGS Publications Warehouse

Ruth, Janet M.; Skagen, Susan K.

2017-01-01

Grassland bird populations are showing some of the greatest rates of decline of any North American birds, prompting measures to protect and improve important habitat. We assessed how vegetation structure and composition, habitat features often targeted for management, affected territory and nest site selection by Grasshopper Sparrows (Ammodramus savannarum ammolegus) in southeastern Arizona. To identify features important to males establishing territories, we compared vegetation characteristics of known territories and random samples on 2 sites over 5 years. We examined habitat selection patterns of females by comparing characteristics of nest sites with territories over 3 years. Males selected territories in areas of sparser vegetation structure and more tall shrubs (>2 m) than random plots on the site with low shrub densities. Males did not select territories based on the proportion of exotic grasses. Females generally located nest sites in areas with lower small shrub (1–2 m tall) densities than territories overall when possible and preferentially selected native grasses for nest construction. Whether habitat selection was apparent depended upon the range of vegetation structure that was available. We identified an upper threshold above which grass structure seemed to be too high and dense for Grasshopper Sparrows. Our results suggest that some management that reduces vegetative structure may benefit this species in desert grasslands at the nest and territory scale. However, we did not assess initial male habitat selection at a broader landscape scale where their selection patterns may be different and could be influenced by vegetation density and structure outside the range of values sampled in this study.

Crystal structure of glucose isomerase in complex with xylitol inhibitor in one metal binding mode.

PubMed

Bae, Ji-Eun; Kim, In Jung; Nam, Ki Hyun

2017-11-04

Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O 2 and O 4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Insights into intermolecular interactions, electrostatic properties and the stability of C646 in the binding pocket of p300 histone acetyltransferase enzyme: a combined molecular dynamics and charge density study.

PubMed

Sivanandam, Magudeeswaran; Saravanan, Kandasamy; Kumaradhas, Poomani

2017-10-30

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are enzymes that exhibit an important transcription activity. Dysfunction of these enzymes may lead to different diseases including cancer, cardiovascular, and other diseases. Therefore, these enzymes are the potential target for the generation of new therapeutics. C646 is a synthetic p300 HAT inhibitor; its structural and the electrostatic properties are the paradigm to understand its activity in the active site of p300 HAT enzyme. The docked C646 molecule in the active site forms expected key intermolecular interactions with the amino acid residues Trp1436, Tyr1467, and one water molecule (W1861); and these interactions are important for acetylation reaction. When compare the active site structure of C646 with the gas-phase structure, it is confirmed that the electron density distribution of polar bonds are highly altered, when the molecule present in the active site. In the gas-phase structure of C646, a large negative regions of electrostatic potential is found at the vicinity of O(4), O(5), and O(6) atoms; whereas, the negative region of these atoms are reduced in the active site. The molecular dynamics (MD) simulation also performed, it reveals the conformational stability and the intermolecular interactions of C646 molecule in the active site of p300.

Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

PubMed

Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

2016-08-01

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Risky business: Site selection by Acadian Flycatchers under threat of nest predation and brood parasitism

USGS Publications Warehouse

HazIer, K.R.; Cooper, R.J.; Twedt, D.J.

2006-01-01

Habitat quality is determined not only by habitat structure and the availability of resources, but also by competitors, cooperators, predators, and parasites. We hypothesized that, for passerines, minimizing risk from avian nest predators and brood parasites is an important factor in selecting a breeding site. Through the early part of two breeding seasons, we spot-mapped locations of Acadian Flycatchers (Empidonax virescens, territory selectors), Red-bellied Woodpeckers (Melanerpes carolinus, nest predators) and Brown-headed Cowbirds (Molothrus ater, brood parasites) in a 56-ha study area within an extensive bottomland hardwood forest. We were thereby able to determine the order of flycatcher territory settlement and nest initiation in relation to risk of predation and parasitism, while accounting for habitat structure. Male settlement was influenced by both habitat structure and risk avoidance. However, risk trom woodpeckers was relatively more important in the first season and risk from cowbirds in the second, evidently due to differences in the relative abundance of predator and brood-parasite in each year. For male flycatchers, settlement choices appear to be flexible in the face of changing 'risk landscapes.' For females, habitat structure was the most important predictor of nest site selection. Even so, there was evidence that females avoided cowbirds. Surprisingly, nest site selection was positively associated with woodpecker abundance in the first season when woodpeckers were present in greater numbers. Possible explanations for this contradictory result are discussed.

Allosteric binding sites in Rab11 for potential drug candidates

PubMed Central

2018-01-01

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously. PMID:29874286

Exploration of enzyme-ligand interactions in CYP2D6 & 3A4 homology models and crystal structures using a novel computational approach.

PubMed

Kjellander, Britta; Masimirembwa, Collen M; Zamora, Ismael

2007-01-01

New crystal structures of human CYP2D6 and CYP3A4 have recently been reported, and in this study, we wanted to compare them with previously used homology models with respect to predictions of site of metabolism and ligand-enzyme interactions. The data set consisted of a family of synthetic opioid analgesics with the aim to cover both CYP2D6 and CYP3A4, as most of these compounds are metabolized by both isoforms. The program MetaSite was used for the site of metabolism predictions, and the results were validated by experimental assessment of the major metabolites formed with recombinant CYP450s. This was made on a selection of 14 compounds in the data set. The prediction rates for MetaSite were 79-100% except for the CYP3A4 homology model, which picked the correct site in half of the cases. Despite differences in orientation of some important amino acids in the active sites, the MetaSite-predicted sites were the same for the different structures, with the exception of the CYP3A4 homology model. Further exploration of interactions with ligands was done by docking substrates/inhibitors in the different structures with the docking program GLUE. To address the challenge in interpreting patterns of enzyme-ligand interactions for the large number of different docking poses, a new computational tool to handle the results from the dockings was developed, in which the output highlights the relative importance of amino acids in CYP450-substrate/inhibitor interactions. The method is based on calculations of the interaction energies for each pose with the surrounding amino acids. For the CYP3A4 structures, this method was compared with consensus principal component analysis (CPCA), a commonly used method for structural comparison to evaluate the usefulness of the new method. The results from the two methods were comparable with each other, and the highlighted amino acids resemble those that were identified to have a different orientation in the compared structures. The new method has clear advantages over CPCA in that it is far simpler to interpret and there is no need for protein alignment. The methodology enables structural comparison but also gives insights on important amino acid substrate/inhibitor interactions and can therefore be very useful when suggesting modifications of new chemical entities to improve their metabolic profiles.

Influence of habitat heterogeneity on the community structure of deep-sea harpacticoid communities from a canyon and an escarpment site on the continental rise off California

NASA Astrophysics Data System (ADS)

Thistle, David; Sedlacek, Linda; Carman, Kevin R.; Barry, James P.

2017-05-01

The sediment-covered deep-sea floor was initially thought to be environmentally homogeneous. Recent work has shown otherwise, and deep-sea ecologists have been searching for ecologically important environmental heterogeneities on different spatial and temporal scales, with particular interest in canyons. Here we report results for harpacticoid copepods from a site at 3262 m depth in the axis of Monterey Canyon and one on an escarpment 46 km away at 3090 m depth. Multivariate community analyses revealed significant differences between sites in community structure. Absolute abundance, the ratio of subadult copepodites to adults, species density, the proportion of the harpacticoid individuals that emerged, and the proportion that lived in tubes were significantly lower at the canyon site than at the escarpment site. The proportion of the harpacticoid individuals that belonged to the surface-dweller life-style group was significantly higher than at the escarpment site. These marked differences imply that ecologically important environmental heterogeneities exist. We speculate that differences between the sites in food conditions and sediment grain-size distributions are among them.

Structural and functional dissection reveals distinct roles of Ca2+-binding sites in the giant adhesin SiiE of Salmonella enterica

PubMed Central

Klingl, Stefan; Sandmann, Achim; Taccardi, Nicola; Sticht, Heinrich; Muller, Yves A.; Hensel, Michael

2017-01-01

The giant non-fimbrial adhesin SiiE of Salmonella enterica mediates the first contact to the apical site of epithelial cells and enables subsequent invasion. SiiE is a 595 kDa protein composed of 53 repetitive bacterial immunoglobulin (BIg) domains and the only known substrate of the SPI4-encoded type 1 secretion system (T1SS). The crystal structure of BIg50-52 of SiiE revealed two distinct Ca2+-binding sites per BIg domain formed by conserved aspartate or glutamate residues. In a mutational analysis Ca2+-binding sites were disrupted by aspartate to serine exchange at various positions in the BIg domains of SiiE. Amounts of secreted SiiE diminish with a decreasing number of intact Ca2+-binding sites. BIg domains of SiiE contain distinct Ca2+-binding sites, with type I sites being similar to other T1SS-secreted proteins and type II sites newly identified in SiiE. We functionally and structurally dissected the roles of type I and type II Ca2+-binding sites in SiiE, as well as the importance of Ca2+-binding sites in various positions of SiiE. Type I Ca2+-binding sites were critical for efficient secretion of SiiE and a decreasing number of type I sites correlated with reduced secretion. Type II sites were less important for secretion, stability and surface expression of SiiE, however integrity of type II sites in the C-terminal portion was required for the function of SiiE in mediating adhesion and invasion. PMID:28558023

Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

PubMed

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

2014-02-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

Evidence of Pervasive Biologically Functional Secondary Structures within the Genomes of Eukaryotic Single-Stranded DNA Viruses

PubMed Central

Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y. F.; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie

2014-01-01

Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here. PMID:24284329

Virus-like attachment sites as structural landmarks of plants retrotransposons.

PubMed

Ochoa Cruz, Edgar Andres; Cruz, Guilherme Marcello Queiroga; Vieira, Andréia Prata; Van Sluys, Marie-Anne

2016-01-01

The genomic data available nowadays has enabled the study of repetitive sequences and their relationship to viruses. Among them, long terminal repeat retrotransposons (LTR-RTs) are the largest component of most plant genomes, the Gypsy and Copia superfamilies being the most common. Recently it has been found that Del lineage, an LTR-RT of Gypsy superfamily, has putative virus-like attachment (vl-att) sites. This signature, originally described for retroviruses, is recognized by retroviral integrase conferring specificity to the integration process. Here we retrieved 26,092 putative complete LTR-RTs from 10 lineages found in 10 fully sequenced angiosperm genomes and found putative vl-att sites that are a conserved structural landmark across these genomes. Furthermore, we reveal that each plant genome has a distinguishable LTR-RT lineage amplification pattern that could be related to the vl-att sites diversity. We used these patterns to generate a specific quick-response (QR) code for each genome that could be used as a barcode of identification of plants in the future. The universal distribution of vl-att sites represents a new structural feature common to plant LTR-RTs and retroviruses. This is an important finding that expands the information about the structural similarity between LTR-RT and retroviruses. We speculate that the sequence diversity of vl-att sites could be important for the life cycle of retrotransposons, as it was shown for retroviruses. All the structural vl-att site signatures are strong candidates for further functional studies. Moreover, this is the first identification of specific LTR-RT content and their amplification patterns in a large dataset of LTR-RT lineages and angiosperm genomes. These distribution patterns could be used in the future with biotechnological identification purposes.

Fold independent structural comparisons of protein-ligand binding sites for exploring functional relationships.

PubMed

Gold, Nicola D; Jackson, Richard M

2006-02-03

The rapid growth in protein structural data and the emergence of structural genomics projects have increased the need for automatic structure analysis and tools for function prediction. Small molecule recognition is critical to the function of many proteins; therefore, determination of ligand binding site similarity is important for understanding ligand interactions and may allow their functional classification. Here, we present a binding sites database (SitesBase) that given a known protein-ligand binding site allows rapid retrieval of other binding sites with similar structure independent of overall sequence or fold similarity. However, each match is also annotated with sequence similarity and fold information to aid interpretation of structure and functional similarity. Similarity in ligand binding sites can indicate common binding modes and recognition of similar molecules, allowing potential inference of function for an uncharacterised protein or providing additional evidence of common function where sequence or fold similarity is already known. Alternatively, the resource can provide valuable information for detailed studies of molecular recognition including structure-based ligand design and in understanding ligand cross-reactivity. Here, we show examples of atomic similarity between superfamily or more distant fold relatives as well as between seemingly unrelated proteins. Assignment of unclassified proteins to structural superfamiles is also undertaken and in most cases substantiates assignments made using sequence similarity. Correct assignment is also possible where sequence similarity fails to find significant matches, illustrating the potential use of binding site comparisons for newly determined proteins.

Changing concepts of geologic structure and the problem of siting nuclear reactors: Examples from Washington State

NASA Astrophysics Data System (ADS)

Tabor, R. W.

1986-09-01

The conflict between regulation and healthy evolution of geological science has contributed to the difficulties of siting nuclear reactors. On the Columbia Plateau in Washington, but for conservative design of the Hanford reactor facility, the recognition of the little-understood Olympic-Wallowa lineament as a major, possibly still active structural alinement might have jeopardized the acceptability of the site for nuclear reactors. On the Olympic Peninsula, evolving concepts of compressive structures and their possible recent activity and the current recognition of a subducting Juan de Fuca plate and its potential for generating great earthquakes—both concepts little-considered during initial site selection—may delay final acceptance of the Satsop site. Conflicts of this sort are inevitable but can be accommodated if they are anticipated in the reactor-licensing process. More important, society should be increasing its store of geologic knowledge now, during the current recess in nuclear reactor siting.

Effects of common seagrass restoration methods on ecosystem structure in subtropical seagrass meadows.

PubMed

Bourque, Amanda S; Fourqurean, James W

2014-06-01

Seagrass meadows near population centers are subject to frequent disturbance from vessel groundings. Common seagrass restoration methods include filling excavations and applying fertilizer to encourage seagrass recruitment. We sampled macrophytes, soil structure, and macroinvertebrate infauna at unrestored and recently restored vessel grounding disturbances to evaluate the effects of these restoration methods on seagrass ecosystem structure. After a year of observations comparing filled sites to both undisturbed reference and unrestored disturbed sites, filled sites had low organic matter content, nutrient pools, and primary producer abundance. Adding a nutrient source increased porewater nutrient pools at disturbed sites and in undisturbed meadows, but not at filled sites. Environmental predictors of infaunal community structure across treatments included soil texture and nutrient pools. At the one year time scale, the restoration methods studied did not result in convergence between restored and unrestored sites. Particularly in filled sites, soil conditions may combine to constrain rapid development of the seagrass community and associated infauna. Our study is important for understanding early recovery trajectories following restoration using these methods. Published by Elsevier Ltd.

X-ray Crystallographic Analysis of [alpha]-Ketoheterocycle Inhibitors Bound to a Humanized Variant of Fatty Acid Amide Hydrolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mileni, Mauro; Garfunkle, Joie; Ezzili, Cyrine

2010-11-03

Three cocrystal X-ray structures of the {alpha}-ketoheterocycle inhibitors 3-5 bound to a humanized variant of fatty acid amide hydrolase (FAAH) are disclosed and comparatively discussed alongside those of 1 (OL-135) and its isomer 2. These five X-ray structures systematically probe each of the three active site regions key to substrate or inhibitor binding: (1) the conformationally mobile acyl chain-binding pocket and membrane access channel responsible for fatty acid amide substrate and inhibitor acyl chain binding, (2) the atypical active site catalytic residues and surrounding oxyanion hole that covalently binds the core of the {alpha}-ketoheterocycle inhibitors captured as deprotonated hemiketals mimickingmore » the tetrahedral intermediate of the enzyme-catalyzed reaction, and (3) the cytosolic port and its uniquely important imbedded ordered water molecules and a newly identified anion binding site. The detailed analysis of their key active site interactions and their implications on the interpretation of the available structure-activity relationships are discussed providing important insights for future design.« less

Exploring Protein Structure and Dynamics through a Project-Oriented Biochemistry Laboratory Module

ERIC Educational Resources Information Center

Lipchock, James M.; Ginther, Patrick S.; Douglas, Bonnie B.; Bird, Kelly E.; Loria, J. Patrick

2017-01-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant…

Predicting Ligand Binding Sites on Protein Surfaces by 3-Dimensional Probability Density Distributions of Interacting Atoms

PubMed Central

Jian, Jhih-Wei; Elumalai, Pavadai; Pitti, Thejkiran; Wu, Chih Yuan; Tsai, Keng-Chang; Chang, Jeng-Yih; Peng, Hung-Pin; Yang, An-Suei

2016-01-01

Predicting ligand binding sites (LBSs) on protein structures, which are obtained either from experimental or computational methods, is a useful first step in functional annotation or structure-based drug design for the protein structures. In this work, the structure-based machine learning algorithm ISMBLab-LIG was developed to predict LBSs on protein surfaces with input attributes derived from the three-dimensional probability density maps of interacting atoms, which were reconstructed on the query protein surfaces and were relatively insensitive to local conformational variations of the tentative ligand binding sites. The prediction accuracy of the ISMBLab-LIG predictors is comparable to that of the best LBS predictors benchmarked on several well-established testing datasets. More importantly, the ISMBLab-LIG algorithm has substantial tolerance to the prediction uncertainties of computationally derived protein structure models. As such, the method is particularly useful for predicting LBSs not only on experimental protein structures without known LBS templates in the database but also on computationally predicted model protein structures with structural uncertainties in the tentative ligand binding sites. PMID:27513851

Text Mining Improves Prediction of Protein Functional Sites

PubMed Central

Cohn, Judith D.; Ravikumar, Komandur E.

2012-01-01

We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

The Wood and Bark of Hardwoods Growing on Southern Pine Sites - A Pictorial Atlas

Treesearch

Charles W. McMillin; Floyd G. Manwiller

1980-01-01

Provides a pictorial description of the structure and appearance of 23 pine-site hardwoods, an overview of hardwood anatomy, and data on the resource and certain important physical properties of stemwood and bark.

Investigations of fungal diversity in wooden structures and soils at historic sites on the Antarctic Peninsula.

PubMed

Arenz, Brett E; Blanchette, Robert A

2009-01-01

Investigations of microbial diversity in Antarctic are important to begin to understand ecosystem functioning and decomposition processes. This study documents fungi at 9 historic sites on the Antarctic Peninsula collected from wooden structures, other organic materials, and soils during a joint National Science Foundation and British Antarctic Survey expedition in 2007. Many of these sites had wooden structures built by the British during the World War II Operation Tabarin, but others visited included the American "East Base" on Stonington Island and the Swedish hut on Snow Hill Island. Fungi were cultured on several different media and pure cultures were obtained and identified by DNA sequencing of the internal transcribed spacer region. Cadophora species previously found to attack historic wooden structures on Ross Island, Antarctica, were found at all but 1 location sampled in the Peninsula region. Fungi causing decay in the historic wooden structures and artifacts and those causing mold problems inside the structures are of great concern, and conservation efforts are urgently needed to help preserve these important polar heritage structures. The results presented also expand our knowledge on the identity of fungi present throughout the Antarctic Peninsula region and provide insights into the organisms responsible for decomposition and nutrient recycling.

Phosphoenolpyruvate carboxylase: a new era of structural biology.

PubMed

Izui, Katsura; Matsumura, Hiroyoshi; Furumoto, Tsuyoshi; Kai, Yasushi

2004-01-01

There have been remarkable advances in our knowledge of this important enzyme in the last decade. This review focuses on three recent topics: the three-dimensional structure of the protein, molecular mechanisms of catalytic and regulatory functions, and the molecular cloning and characterization of PEPC kinases, which are Ser/Thr kinases involved specifically in regulatory phosphorylation of vascular plant PEPC. Analysis by X-ray crystallography and site-directed mutagenesis for E. coli and maize PEPC identified the catalytic site and allosteric effector binding sites, and revealed the functional importance of mobile loops. We present the reaction mechanism of PEPC in which we assign the roles of individual amino acid residues. We discuss the unique molecular property of PEPC kinase and its possible regulation at the post-translational level.

Prediction of enzyme binding: human thrombin inhibition study by quantum chemical and artificial intelligence methods based on X-ray structures.

PubMed

Mlinsek, G; Novic, M; Hodoscek, M; Solmajer, T

2001-01-01

Thrombin is a serine protease which plays important roles in the human body, the key one being the control of thrombus formation. The inhibition of thrombin has become a target for new antithrombotics. The aim of our work was to (i) construct a model which would enable us to predict Ki values for the binding of an inhibitor into the active site of thrombin based on a database of known X-ray structures of inhibitor-enzyme complexes and (ii) to identify the structural and electrostatic characteristics of inhibitor molecules crucially important to their effective binding. To retain as much of the 3D structural information of the bound inhibitor as possible, we implemented the quantum mechanical/molecular mechanical (QM/MM) procedure for calculating the molecular electrostatic potential (MEP) at the van der Waals surfaces of atoms in the protein's active site. The inhibitor was treated quantum mechanically, while the rest of the complex was treated by classical means. The obtained MEP values served as inputs into the counter-propagation artificial neural network (CP-ANN), and a genetic algorithm was subsequently used to search for the combination of atoms that predominantly influences the binding. The constructed CP-ANN model yielded Ki values predictions with a correlation coefficient of 0.96, with Ki values extended over 7 orders of magnitude. Our approach also shows the relative importance of the various amino acid residues present in the active site of the enzyme for inhibitor binding. The list of residues selected by our automatic procedure is in good correlation with the current consensus regarding the importance of certain crucial residues in thrombin's active site.

Influence of fine-scale habitat structure on nest-site occupancy, laying date and clutch size in Blue Tits Cyanistes caeruleus

NASA Astrophysics Data System (ADS)

Amininasab, Seyed Mehdi; Vedder, Oscar; Schut, Elske; de Jong, Berber; Magrath, Michael J. L.; Korsten, Peter; Komdeur, Jan

2016-01-01

Most birds have specific habitat requirements for breeding. The vegetation structure surrounding nest-sites is an important component of habitat quality, and can have large effects on avian breeding performance. We studied 13 years of Blue Tit Cyanistes caeruleus population data to determine whether characteristics of vegetation structure predict site occupancy, laying date and number of eggs laid. Measurements of vegetation structure included the density of English Oak Quercus robur, European Beech Fagus sylvatica, and other deciduous, coniferous and non-coniferous evergreen trees, within a 20-m radius of nest-boxes used for breeding. Trees were further sub-divided into specific classes of trunk circumferences to determine the densities for different maturity levels. Based on Principal Component Analysis (PCA), we reduced the total number of 17 measured vegetation variables to 7 main categories, which we used for further analyses. We found that the occupancy rate of sites and the number of eggs laid correlated positively with the proportion of deciduous trees and negatively with the density of coniferous trees. Laying of the first egg was advanced with a greater proportion of deciduous trees. Among deciduous trees, the English Oak appeared to be most important, as a higher density of more mature English Oak trees was associated with more frequent nest-box occupancy, a larger number of eggs laid, and an earlier laying start. Furthermore, laying started earlier and more eggs were laid in nest-boxes with higher occupancy rates. Together, these findings highlight the role of deciduous trees, particularly more mature English Oak, as important predictors of high-quality preferred habitat. These results aid in defining habitat quality and will facilitate future studies on the importance of environmental quality for breeding performance.

CCProf: exploring conformational change profile of proteins

PubMed Central

Chang, Che-Wei; Chou, Chai-Wei; Chang, Darby Tien-Hao

2016-01-01

In many biological processes, proteins have important interactions with various molecules such as proteins, ions or ligands. Many proteins undergo conformational changes upon these interactions, where regions with large conformational changes are critical to the interactions. This work presents the CCProf platform, which provides conformational changes of entire proteins, named conformational change profile (CCP) in the context. CCProf aims to be a platform where users can study potential causes of novel conformational changes. It provides 10 biological features, including conformational change, potential binding target site, secondary structure, conservation, disorder propensity, hydropathy propensity, sequence domain, structural domain, phosphorylation site and catalytic site. All these information are integrated into a well-aligned view, so that researchers can capture important relevance between different biological features visually. The CCProf contains 986 187 protein structure pairs for 3123 proteins. In addition, CCProf provides a 3D view in which users can see the protein structures before and after conformational changes as well as binding targets that induce conformational changes. All information (e.g. CCP, binding targets and protein structures) shown in CCProf, including intermediate data are available for download to expedite further analyses. Database URL: http://zoro.ee.ncku.edu.tw/ccprof/ PMID:27016699

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.

The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn{sub 0.55}{sup 2+}Fe{sub 0.18}{sup 3+}){sub tet}[Zr{sub 0.45}{sup 2+}Fe{sub 1.82}{sup 3+}]{sub oct}O{sub 4} through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zincmore » cations, but not the iron cations, with respect to the spinel structure. The presence of Fe{sup 3+} on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics.« less

Early Deterioration of Coarse Woody Debris

Treesearch

F.H. Tainter; J.W. McMinn

1999-01-01

Coarse woody debris (CWD) is an important structural component of southern forest ecosystems. CWD loading may be affected by different decomposition rates on sites of varying quality. Bolts of red oak and loblolly pine were placed on plots at each of three (hydric, mesic. and xeric) sites at the Savannah River Site and sampled over a 16-week period. Major changes...

Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

PubMed

Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

2018-04-03

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eren, Elif; Murphy, Megan; Goguen, Jon

2010-08-13

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changesmore » of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.« less

Open challenges in structure-based virtual screening: Receptor modeling, target flexibility consideration and active site water molecules description.

PubMed

Spyrakis, Francesca; Cavasotto, Claudio N

2015-10-01

Structure-based virtual screening is currently an established tool in drug lead discovery projects. Although in the last years the field saw an impressive progress in terms of algorithm development, computational performance, and retrospective and prospective applications in ligand identification, there are still long-standing challenges where further improvement is needed. In this review, we consider the conceptual frame, state-of-the-art and recent developments of three critical "structural" issues in structure-based drug lead discovery: the use of homology modeling to accurately model the binding site when no experimental structures are available, the necessity of accounting for the dynamics of intrinsically flexible systems as proteins, and the importance of considering active site water molecules in lead identification and optimization campaigns. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of Conserved Water Sites in Protein Structures for Drug Design.

PubMed

Jukič, Marko; Konc, Janez; Gobec, Stanislav; Janežič, Dušanka

2017-12-26

Identification of conserved waters in protein structures is a challenging task with applications in molecular docking and protein stability prediction. As an alternative to computationally demanding simulations of proteins in water, experimental cocrystallized waters in the Protein Data Bank (PDB) in combination with a local structure alignment algorithm can be used for reliable prediction of conserved water sites. We developed the ProBiS H2O approach based on the previously developed ProBiS algorithm, which enables identification of conserved water sites in proteins using experimental protein structures from the PDB or a set of custom protein structures available to the user. With a protein structure, a binding site, or an individual water molecule as a query, ProBiS H2O collects similar proteins from the PDB and performs local or binding site-specific superimpositions of the query structure with similar proteins using the ProBiS algorithm. It collects the experimental water molecules from the similar proteins and transposes them to the query protein. Transposed waters are clustered by their mutual proximity, which enables identification of discrete sites in the query protein with high water conservation. ProBiS H2O is a robust and fast new approach that uses existing experimental structural data to identify conserved water sites on the interfaces of protein complexes, for example protein-small molecule interfaces, and elsewhere on the protein structures. It has been successfully validated in several reported proteins in which conserved water molecules were found to play an important role in ligand binding with applications in drug design.

Domain alternation and active site remodeling are conserved structural features of ubiquitin E1.

PubMed

Lv, Zongyang; Yuan, Lingmin; Atkison, James H; Aldana-Masangkay, Grace; Chen, Yuan; Olsen, Shaun K

2017-07-21

E1 enzymes for ubiquitin (Ub) and Ub-like modifiers (Ubls) harbor two catalytic activities that are required for Ub/Ubl activation: adenylation and thioester bond formation. Structural studies of the E1 for the Ubl s mall u biquitin-like mo difier (SUMO) revealed a single active site that is transformed by a conformational switch that toggles its competency for catalysis of these two distinct chemical reactions. Although the mechanisms of adenylation and thioester bond formation revealed by SUMO E1 structures are thought to be conserved in Ub E1, there is currently a lack of structural data supporting this hypothesis. Here, we present a structure of Schizosaccharomyces pombe Uba1 in which the second catalytic cysteine half-domain (SCCH domain) harboring the catalytic cysteine has undergone a 106° rotation that results in a completely different network of intramolecular interactions between the SCCH and adenylation domains and translocation of the catalytic cysteine 12 Å closer to the Ub C terminus compared with previous Uba1 structures. SCCH domain alternation is accompanied by conformational changes within the Uba1 adenylation domains that effectively disassemble the adenylation active site. Importantly, the structural and biochemical data suggest that domain alternation and remodeling of the adenylation active site are interconnected and are intrinsic structural features of Uba1 and that the overall structural basis for adenylation and thioester bond formation exhibited by SUMO E1 is indeed conserved in Ub E1. Finally, the mechanistic insights provided by the novel conformational snapshot of Uba1 presented in this study may guide efforts to develop small molecule inhibitors of this critically important enzyme that is an active target for anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Lipid Interaction Sites on Channels, Transporters and Receptors: Recent Insights from Molecular Dynamics Simulations

PubMed Central

Hedger, George; Sansom, Mark S. P.

2017-01-01

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterisation of these sites is of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. PMID:26946244

The N Domain of Human Angiotensin-I-converting Enzyme

PubMed Central

Anthony, Colin S.; Corradi, Hazel R.; Schwager, Sylva L. U.; Redelinghuys, Pierre; Georgiadis, Dimitris; Dive, Vincent; Acharya, K. Ravi; Sturrock, Edward D.

2010-01-01

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding. PMID:20826823

Determination of the Basin Structure Beneath European Side of Istanbul

NASA Astrophysics Data System (ADS)

Karabulut, Savas; Cengiz Cinku, Mulla; Thomas, Michael; Lamontagne, Maurice

2016-04-01

Istanbul (near North Anatolian Fault Zone:NAFZ, Turkey) is located in northern part of Sea of Marmara, an area that has been influenced by possible Marmara Earthquakes. The general geology of Istanbul divided into two stratigraphic unit such as sedimentary (from Oligocene to Quaternary Deposits) and bedrock (Paleozoic and Eocene). The bedrock units consists of sand stone, clay stone to Paleozoic age and limestone to Eocene age and sedimentary unit consist of sand, clay, mil and gravel from Oligocene to Quaternary age. Earthquake disaster mitigation studies divided into two important phases, too. Firstly, earthquake, soil and engineering structure problems identify for investigation area, later on strategic emergency plan can prepare for these problems. Soil amplification play important role the disaster mitigation and the site effect analysis and basin structure is also a key parameter for determining of site effect. Some geophysical, geological and geotechnical measurements are requeired to defined this relationship. Istanbul Megacity has been waiting possible Marmara Earthquake and their related results. In order to defined to possible damage potential related to site effect, gravity measurements carried out for determining to geological structure, basin geometry and faults in Istanbul. Gravity data were collected at 640 sites by using a Scientrex CG-5 Autogravity meter Standard corrections applied to the gravity data include those for instrumental drift, Earth tides and latitude, and the free-air and Bouguer corrections. The corrected gravity data were imported into a Geosoft database to create a grid and map of the Bouguer gravity anomaly (grid cell size of 200 m). As a previously results, we determined some lineminants, faults and basins beneath Istanbul City. Especially, orientation of faults were NW-SE direction and some basin structures determined on between Buyukcekmece and Kucukcekmece Lake.

sc-PDB: an annotated database of druggable binding sites from the Protein Data Bank.

PubMed

Kellenberger, Esther; Muller, Pascal; Schalon, Claire; Bret, Guillaume; Foata, Nicolas; Rognan, Didier

2006-01-01

The sc-PDB is a collection of 6 415 three-dimensional structures of binding sites found in the Protein Data Bank (PDB). Binding sites were extracted from all high-resolution crystal structures in which a complex between a protein cavity and a small-molecular-weight ligand could be identified. Importantly, ligands are considered from a pharmacological and not a structural point of view. Therefore, solvents, detergents, and most metal ions are not stored in the sc-PDB. Ligands are classified into four main categories: nucleotides (< 4-mer), peptides (< 9-mer), cofactors, and organic compounds. The corresponding binding site is formed by all protein residues (including amino acids, cofactors, and important metal ions) with at least one atom within 6.5 angstroms of any ligand atom. The database was carefully annotated by browsing several protein databases (PDB, UniProt, and GO) and storing, for every sc-PDB entry, the following features: protein name, function, source, domain and mutations, ligand name, and structure. The repository of ligands has also been archived by diversity analysis of molecular scaffolds, and several chemoinformatics descriptors were computed to better understand the chemical space covered by stored ligands. The sc-PDB may be used for several purposes: (i) screening a collection of binding sites for predicting the most likely target(s) of any ligand, (ii) analyzing the molecular similarity between different cavities, and (iii) deriving rules that describe the relationship between ligand pharmacophoric points and active-site properties. The database is periodically updated and accessible on the web at http://bioinfo-pharma.u-strasbg.fr/scPDB/.

OnTheFly: a database of Drosophila melanogaster transcription factors and their binding sites.

PubMed

Shazman, Shula; Lee, Hunjoong; Socol, Yakov; Mann, Richard S; Honig, Barry

2014-01-01

We present OnTheFly (http://bhapp.c2b2.columbia.edu/OnTheFly/index.php), a database comprising a systematic collection of transcription factors (TFs) of Drosophila melanogaster and their DNA-binding sites. TFs predicted in the Drosophila melanogaster genome are annotated and classified and their structures, obtained via experiment or homology models, are provided. All known preferred TF DNA-binding sites obtained from the B1H, DNase I and SELEX methodologies are presented. DNA shape parameters predicted for these sites are obtained from a high throughput server or from crystal structures of protein-DNA complexes where available. An important feature of the database is that all DNA-binding domains and their binding sites are fully annotated in a eukaryote using structural criteria and evolutionary homology. OnTheFly thus provides a comprehensive view of TFs and their binding sites that will be a valuable resource for deciphering non-coding regulatory DNA.

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen

2010-04-26

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, themore » streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.« less

Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

PubMed

Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2017-09-01

Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Variation in species diversity and functional traits of sponge communities near human populations in Bocas del Toro, Panama

PubMed Central

Matterson, Kenan O.; Freeman, Christopher J.; Archer, Stephanie K.; Thacker, Robert W.

2015-01-01

Recent studies have renewed interest in sponge ecology by emphasizing the functional importance of sponges in a broad array of ecosystem services. Many critically important habitats occupied by sponges face chronic stressors that might lead to alterations in their diversity, relatedness, and functional attributes. We addressed whether proximity to human activity might be a significant factor in structuring sponge community composition, as well as potential functional roles, by monitoring sponge diversity and abundance at two structurally similar sites that vary in distance to areas of high coastal development in Bocas Del Toro, Panama. We surveyed sponge communities at each site using belt transects and differences between two sites were compared using the following variables: (1) sponge species richness, Shannon diversity, and inverse Simpson’s diversity; (2) phylogenetic diversity; (3) taxonomic and phylogenetic beta diversity; (4) trait diversity and dissimilarity; and (5) phylogenetic and trait patterns in community structure. We observed significantly higher sponge diversity at Punta Caracol, the site most distant from human development (∼5 km). Although phylogenetic diversity was lower at Saigon Bay, the site adjacent to a large village including many houses, businesses, and an airport, the sites did not exhibit significantly different patterns of phylogenetic relatedness in species composition. However, each site had a distinct taxonomic and phylogenetic composition (beta diversity). In addition, the sponge community at Saigon included a higher relative abundance of sponges with high microbial abundance and high chlorophyll a concentration, whereas the community at Punta Caracol had a more even distribution of these traits, yielding a significant difference in functional trait diversity between sites. These results suggest that lower diversity and potentially altered community function might be associated with proximity to human populations. This study highlights the importance of evaluating functional traits and phylogenetic diversity in addition to common diversity metrics when assessing potential environmental impacts on benthic communities. PMID:26587347

Engaging, Recruiting, and Retaining Black Men Who Have Sex With Men in Research Studies: Don’t Underestimate the Importance of Staffing—Lessons Learned From HPTN 061, the BROTHERS Study

PubMed Central

Magnus, Manya; Franks, Julie; Griffith, Sam; Arnold, Michael P.; Goodman, Krista; Wheeler, Darrell P.

2014-01-01

Context HIV/AIDS in the United States continues to primarily impact men who have sex with men (MSM), with disproportionately high rates among black MSM. Objective The purpose of this study was to identify factors that may influence engagement and retention of black MSM in HIV research. Design and Participants This was a qualitative evaluation of study implementation within a multisite, prospective, observational study (HIV Prevention Trials Network 061, BROTHERS) that enrolled 1553 black MSM in 6 cities throughout the United States. Data collection for this evaluation included a written, structured survey collected from each of the sites describing site characteristics including staff and organizational structure, reviews of site standard operating procedures, and work plans; semistructured key informant interviews were conducted with site coordinators to characterize staffing, site-level factors facilitating or impeding effective community engagement, study recruitment, and retention. Data from completed surveys and site standard operating procedures were collated, and notes from key informant interviews were thematically coded for content by 2 independent reviewers. Results Several key themes emerged from the data, including the importance of inclusion of members of the community being studied as staff, institutional hiring practices that support inclusive staffing, cultivating a supportive working environment for study implementation, and ongoing relationships between research institutions and community. Conclusions This study underscores the importance of staffing in implementing research with black MSM. Investigators should consider how staffing and organizational structures affect implementation during study design and when preparing to initiate study activities. Ongoing monitoring of community engagement can inform and improve methods for engagement and ensure cultural relevance while removing barriers for participation. PMID:24406940

Engaging, recruiting, and retaining black men who have sex with men in research studies: don't underestimate the importance of staffing--lessons learned from HPTN 061, the BROTHERS study.

PubMed

Magnus, Manya; Franks, Julie; Griffith, Sam; Arnold, Michael P; Goodman, Krista; Wheeler, Darrell P

2014-01-01

HIV/AIDS in the United States continues to primarily impact men who have sex with men (MSM), with disproportionately high rates among black MSM. The purpose of this study was to identify factors that may influence engagement and retention of black MSM in HIV research. This was a qualitative evaluation of study implementation within a multisite, prospective, observational study (HIV Prevention Trials Network 061, BROTHERS) that enrolled 1553 black MSM in 6 cities throughout the United States. Data collection for this evaluation included a written, structured survey collected from each of the sites describing site characteristics including staff and organizational structure, reviews of site standard operating procedures, and work plans; semistructured key informant interviews were conducted with site coordinators to characterize staffing, site-level factors facilitating or impeding effective community engagement, study recruitment, and retention. Data from completed surveys and site standard operating procedures were collated, and notes from key informant interviews were thematically coded for content by 2 independent reviewers. Several key themes emerged from the data, including the importance of inclusion of members of the community being studied as staff, institutional hiring practices that support inclusive staffing, cultivating a supportive working environment for study implementation, and ongoing relationships between research institutions and community. This study underscores the importance of staffing in implementing research with black MSM. Investigators should consider how staffing and organizational structures affect implementation during study design and when preparing to initiate study activities. Ongoing monitoring of community engagement can inform and improve methods for engagement and ensure cultural relevance while removing barriers for participation.

Stand Structural Controls on Evapotranspiration in Native and Invaded Tropical Montane Cloud Forest in Hawai'i

NASA Astrophysics Data System (ADS)

Giambelluca, T. W.; Delay, J. K.; Asner, G. P.; Martin, R. E.; Nullet, M. A.; Huang, M.; Mudd, R. G.; Takahashi, M.

2008-12-01

Tropical montane cloud forests (TMCFs) in Hawai'i are important zones of water input and stores of critically important native plant and animal species. Invasion by alien tree species threatens these forests and may alter the hydrological services they provide. At two TMCF sites in Hawai'i, one within native Metrosideros polymorpha forest and the other at a site heavily invaded by Psidium cattleianum, we are conducting measurements of stand-level evapotranspiration (ET), transpiration (using sapflow techniques), energy balance, and related processes. Previously presented results showed that ET as a function of available energy was 27% higher at the invaded site than the native site, with the difference rising to 53% during dry- canopy periods. In this presentation, mechanisms for the observed higher ET rate at the invaded site are explored. The difference in measured xylem flow velocities of native and alien trees cannot explain the observed stand level ET difference. Tree basal area is lower at the invaded site than the native site, again contrary to the ET difference. However, the alien trees have much smaller stem diameters, on average, than the native trees, with little or no heartwood. Hence, the cross-sectional xylem area is much greater in the invaded stand, facilitating higher transpiration rates. These results demonstrate the importance of stand structural controls on ET and raise questions about whether higher ET is a transient feature of the succession or a persistent characteristic of invasive trees.

Studies of the reactivity of the ferrihydrite surface by iron isotopic exchange and Mossbauer spectroscopy

USGS Publications Warehouse

Rea, Brigid A.; Davis, James A.; Waychunas , Glenn A.

1994-01-01

Two-line ferrihydrite is an important adsorbent of many toxics in natural and anthropogenic systems; however, the specific structural sites responsible for the high adsorption capacity are not well understood. A combination of chemical and spectroscopic techniques have been employed in this study to gain further insight into the structural nature of sites at the ferrihydrite surface. The kinetics of iron isotopic exchange demonstrated that there are at least two types of iron sites in ferrihydrite. One population of sites, referred to as labile sites, approached iron isotopic equilibrium within 24 hr in 59Fe-NTA solutions, while the second population of sites, referred to as non-labile, exhibited a much slower rate of isotopic exchange. Adsorbed arsenate reduced the degree of exchange by labile sites, indicating that the anion blocked or greatly inhibited the rate of exchange of these sites. Mössbauer spectra were collected from a variety of samples including 56Fe-ferrihydrite samples with 57Fe in labile sites, samples containing 57Fe throughout the structure, and samples with 57Fe in non-labile sites. The spectra showed characteristic broad doublets signifying poor structural order. Refined fits of the spectra indicated that labile sites have larger quadrupole splitting, hence more local distortion, than non-labile sites. In all cases, the spectra demonstrated some degree of asymmetry, indicating a distribution of Fe environments in ferrihydrite. Overall spectral findings, combined with recent EXAFS results (Waychunas et al., 1993), indicate that labile sites likely are more reactive (with respect to iron isotopic exchange) because they have fewer neighboring Fe octahedra and are therefore bound less strongly to the ferrihydrite structure. The labile population of sites probably is composed of end sites of the dioctahedral chain structure of 2-line ferrihydrite, which is a subset of the entire population of surface sites. Mössbauer spectra of samples containing adsorbed arsenate indicated that the anion may slightly decrease the distortion of labile sites and stabilized the structure as a whole by bidentate bonding.

Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

PubMed Central

Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

1999-01-01

A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility.

PubMed

Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K; Quach, Helen Ngoc Bao; Joshi, Adita; LaFave, Matthew C; Burgess, Shawn M; Sampath, Karuna

2016-04-07

DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering. Copyright © 2016 Vrljicak et al.

Pulling History from the Waste Stream: Identification and Collection of Manhattan Project and Cold War Era Artifacts on the Hanford Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marceau, Thomas E.; Watson, Thomas L.

One man's trash is another man's treasure. Not everything called "waste" is meant for the refuse pile. The mission of the Curation Program is at direct odds with the remediation objectives of the Hanford Site. While others are busily tearing down and burying the Site's physical structures and their associated contents, the Curation Program seeks to preserve the tangible elements of the Site's history from these structures for future generations before they flow into the waste stream. Under the provisions of a Programmatic Agreement, Cultural Resources staff initiated a project to identify and collect artifacts and archives that have historicmore » or interpretive value in documenting the role of the Hanford Site throughout the Manhattan Project and Cold War Era. The genesis of Hanford's modern day Curation Program, its evolution over nearly two decades, issues encountered, and lessons learned along the way -- particularly the importance of upper management advocacy, when and how identification efforts should be accomplished, the challenges of working within a radiological setting, and the importance of first hand information -- are presented.« less

Carcass enrichment does not alter decay rates or arthropod community structure: a test of the arthropod saturation hypothesis at the anthropology research facility in Knoxville, Tennessee.

PubMed

Shahid, S Adam; Schoenly, Kenneth; Haskell, Neal H; Hall, Robert D; Zhang, Wenjun

2003-07-01

In a test of an arthropod saturation hypothesis, we asked if the 30-yr history of carcass enrichment at the Anthropology Research Facility, Knoxville TN, has altered carcass decay rates or community structure of sarcosaprophagous arthropods, compared with three local nonenriched sites. Over a 12-d period in 1998, using pitfall traps and sweep nets, we sampled a total of 81,000 invertebrates from freshly euthanized pigs (Sus scrofa L.) placed in these sites. From this number, we sorted 69,286 forensically important (sarcosaprophagous) arthropods. The community structure of these organisms, as measured by species and individuals accumulation curves, rarefaction, and nonparametric correlation, was comparable in all four sites in taxonomic similarity, colonization rates, aerial species richness, and ranked abundances of forensically important taxa on a per carcass basis. Measures of carcass decay rate, remaining carcass weight (%) and periodic weight loss, also were similar. In most cases, carcass surface temperatures and maggot mass temperatures were also statistically indistinguishable. Probability-based results and posthoc power analyses of these variables led us to conclude that the sarcosaprophagous arthropod community of the Anthropology Research Facility is representative of surrounding sites.

Solar Photovoltaic Financing: Deployment by Federal Government Agencies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cory, K.; Coggeshall, C.; Coughlin, J.

2009-07-01

The goal of this report is to examine how federal agencies can finance on-site PV projects. It explains state-level cash incentives available, the importance of solar renewable energy certificate revenues (in certain markets), existing financing structures, as well as innovative financing structures being used by federal agencies to deploy on-site PV. Specific examples from the DOD, DOE, and other federal agencies are highlighted to explain federal project financing in detail.

Contrasting patterns of fine-scale herb layer species composition in temperate forests

NASA Astrophysics Data System (ADS)

Chudomelová, Markéta; Zelený, David; Li, Ching-Feng

2017-04-01

Although being well described at the landscape level, patterns in species composition of forest herb layer are rarely studied at smaller scales. Here, we examined fine-scale environmental determinants and spatial structures of herb layer communities in thermophilous oak- and hornbeam dominated forests of the south-eastern part of the Czech Republic. Species composition of herb layer vegetation and environmental variables were recorded within a fixed grid of 2 × 2 m subplots regularly distributed within 1-ha quadrate plots in three forest stands. For each site, environmental models best explaining species composition were constructed using constrained ordination analysis. Spatial eigenvector mapping was used to model and account for spatial structures in community variation. Mean Ellenberg indicator values calculated for each subplot were used for ecological interpretation of spatially structured residual variation. The amount of variation explained by environmental and spatial models as well as the selection of variables with the best explanatory power differed among sites. As an important environmental factor, relative elevation was common to all three sites, while pH and canopy openness were shared by two sites. Both environmental and community variation was mostly coarse-scaled, as was the spatially structured portion of residual variation. When corrected for bias due to spatial autocorrelation, those environmental factors with already weak explanatory power lost their significance. Only a weak evidence of possibly omitted environmental predictor was found for autocorrelated residuals of site models using mean Ellenberg indicator values. Community structure was determined by different factors at different sites. The relative importance of environmental filtering vs. spatial processes was also site specific, implying that results of fine-scale studies tend to be shaped by local conditions. Contrary to expectations based on other studies, overall dominance of spatial processes at fine scale has not been detected. Ecologists should keep this in mind when making generalizations about community dynamics.

Investigating the Surface Structure of γ-Al 2 O 3 Supported WO X Catalysts by High Field 27 Al MAS NMR and Electronic Structure Calculations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Chuan; Hu, Mary Y.; Jaegers, Nicholas R.

The metal-support interaction in γ-Al2O3 supported WOX catalysts is investigated by a combination of high field quantitative single pulse (SP) 27Al MAS NMR spectroscopy, 2D MQMAS, 1H-27Al CP/MAS, and electronic structure calculations. NMR allows the observation of at least seven different Al sites, including a pentahedral Al site, three different tetrahedral Al sites, and three octahedral Al sites. It is found that the penta-coordinated Al (AlP) site density decreases monotonically with an increased WOX loading while the octahedral Al (AlO) site density increases concurrently. This suggests that the Alp sites are the preferred surface anchoring positions for the WOX species.more » Importantly, the AlP site isotropic chemical shift observed for the unsupported γ-Al2O3 at about 38 ppm migrates into the octahedral region with a new isotropic chemical shift value appearing near 7 ppm when the Alp site is anchored by WOX species. Density functional theory (DFT) computational modeling of the NMR parameters on proposed cluster models is carried out to accurately interpret the dramatic chemical shift changes from which the detailed anchoring mechanisms are obtained. It is found that tungsten dimers and monomers are the preferred supported surface species on γ-Al2O3, wherein one monomeric and several dimeric structures are identified as the most likely surface anchoring structures.« less

Pseudomonas aeruginosa 4-Amino-4-Deoxychorismate Lyase: Spatial Conservation of an Active Site Tyrosine and Classification of Two Types of Enzyme

PubMed Central

O'Rourke, Patrick E. F.; Eadsforth, Thomas C.; Fyfe, Paul K.; Shepherd, Sharon M.; Hunter, William N.

2011-01-01

4-Amino-4-deoxychorismate lyase (PabC) catalyzes the formation of 4-aminobenzoate, and release of pyruvate, during folate biosynthesis. This is an essential activity for the growth of Gram-negative bacteria, including important pathogens such as Pseudomonas aeruginosa. A high-resolution (1.75 Å) crystal structure of PabC from P. aeruginosa has been determined, and sequence-structure comparisons with orthologous structures are reported. Residues around the pyridoxal 5′-phosphate cofactor are highly conserved adding support to aspects of a mechanism generic for enzymes carrying that cofactor. However, we suggest that PabC can be classified into two groups depending upon whether an active site and structurally conserved tyrosine is provided from the polypeptide that mainly forms an active site or from the partner subunit in the dimeric assembly. We considered that the conserved tyrosine might indicate a direct role in catalysis: that of providing a proton to reduce the olefin moiety of substrate as pyruvate is released. A threonine had previously been suggested to fulfill such a role prior to our observation of the structurally conserved tyrosine. We have been unable to elucidate an experimentally determined structure of PabC in complex with ligands to inform on mechanism and substrate specificity. Therefore we constructed a computational model of the catalytic intermediate docked into the enzyme active site. The model suggests that the conserved tyrosine helps to create a hydrophobic wall on one side of the active site that provides important interactions to bind the catalytic intermediate. However, this residue does not appear to participate in interactions with the C atom that undergoes an sp 2 to sp 3 conversion as pyruvate is produced. The model and our comparisons rather support the hypothesis that an active site threonine hydroxyl contributes a proton used in the reduction of the substrate methylene to pyruvate methyl in the final stage of the mechanism. PMID:21935381

Near-Seafloor Magnetic Exploration of Submarine Hydrothermal Systems in the Kermadec Arc

NASA Astrophysics Data System (ADS)

Caratori Tontini, F.; de Ronde, C. E. J.; Tivey, M.; Kinsey, J. C.

2014-12-01

Magnetic data can provide important information about hydrothermal systems because hydrothermal alteration can drastically reduce the magnetization of the host volcanic rocks. Near-seafloor data (≤70 m altitude) are required to map hydrothermal systems in detail; Autonomous Underwater Vehicles (AUVs) are the ideal platform to provide this level of resolution. Here, we show the results of high-resolution magnetic surveys by the ABE and Sentry AUVs for selected submarine volcanoes of the Kermadec arc. 3-D magnetization models derived from the inversion of magnetic data, when combined with high resolution seafloor bathymetry derived from multibeam surveys, provide important constraints on the subseafloor geometry of hydrothermal upflow zones and the structural control on the development of seafloor hydrothermal vent sites as well as being a tool for the discovery of previously unknown hydrothermal sites. Significant differences exist between the magnetic expressions of hydrothermal sites at caldera volcanoes ("donut" pattern) and cones ("Swiss cheese" pattern), respectively. Subseafloor 3-D magnetization models also highlight structural differences between focused and diffuse vent sites.

Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

PubMed

Ahmed, Ahmed H; Oswald, Robert E

2010-03-11

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

PubMed Central

Ahmed, Ahmed H.; Oswald, Robert E.

2010-01-01

Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

Structural Explanation for Allolactose (lac Operon Inducer) Synthesis by lacZ β-Galactosidase and the Evolutionary Relationship between Allolactose Synthesis and the lac Repressor

PubMed Central

Wheatley, Robert W.; Lo, Summie; Jancewicz, Larisa J.; Dugdale, Megan L.; Huber, Reuben E.

2013-01-01

β-Galactosidase (lacZ) has bifunctional activity. It hydrolyzes lactose to galactose and glucose and catalyzes the intramolecular isomerization of lactose to allolactose, the lac operon inducer. β-Galactosidase promotes the isomerization by means of an acceptor site that binds glucose after its cleavage from lactose and thus delays its exit from the site. However, because of its relatively low affinity for glucose, details of this site have remained elusive. We present structural data mapping the glucose site based on a substituted enzyme (G794A-β-galactosidase) that traps allolactose. Various lines of evidence indicate that the glucose of the trapped allolactose is in the acceptor position. The evidence includes structures with Bis-Tris (2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol) and l-ribose in the site and kinetic binding studies with substituted β-galactosidases. The site is composed of Asn-102, His-418, Lys-517, Ser-796, Glu-797, and Trp-999. Ser-796 and Glu-797 are part of a loop (residues 795–803) that closes over the active site. This loop appears essential for the bifunctional nature of the enzyme because it helps form the glucose binding site. In addition, because the loop is mobile, glucose binding is transient, allowing the release of some glucose. Bioinformatics studies showed that the residues important for interacting with glucose are only conserved in a subset of related enzymes. Thus, intramolecular isomerization is not a universal feature of β-galactosidases. Genomic analyses indicated that lac repressors were co-selected only within the conserved subset. This shows that the glucose binding site of β-galactosidase played an important role in lac operon evolution. PMID:23486479

Structural analysis of peptides that fill sites near the active center of the two different enzyme molecules by artificial intelligence and computer simulations

NASA Astrophysics Data System (ADS)

Nishiyama, Katsuhiko

2018-05-01

Using artificial intelligence, the binding styles of 167 tetrapeptides were predicted in the active site of papain and cathepsin K. Five tetrapeptides (Asn-Leu-Lys-Trp, Asp-Gln-Trp-Gly, Cys-Gln-Leu-Arg, Gln-Leu-Trp-Thr and Arg-Ser-Glu-Arg) were found to bind sites near the active center of both papain and cathepsin K. These five tetrapeptides have the potential to also bind sites of other cysteine proteases, and structural characteristics of these tetrapeptides should aid the design of a common inhibitor of cysteine proteases. Smart application of artificial intelligence should accelerate data mining of important complex systems.

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

PubMed Central

Schultz, Eric P.; Vasquez, Ernesto E.; Scott, William G.

2014-01-01

The hammerhead ribozyme catalyzes RNA cleavage via acid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK a of the substituted purine; in both cases inosine, which is similar to G in pK a and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data. PMID:25195740

Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid-base catalysis.

PubMed

Schultz, Eric P; Vasquez, Ernesto E; Scott, William G

2014-09-01

The hammerhead ribozyme catalyzes RNA cleavage via acid-base catalysis. Whether it does so by general acid-base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid-base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme from Schistosoma mansoni with a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pK(a) of the substituted purine; in both cases inosine, which is similar to G in pK(a) and hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data.

Ligation site in proteins recognized in silico

PubMed Central

Brylinski, Michal; Konieczny, Leszek; Roterman, Irena

2006-01-01

Recognition of a ligation site in a protein molecule is important for identifying its biological activity. The model for in silico recognition of ligation sites in proteins is presented. The idealized hydrophobic core stabilizing protein structure is represented by a three-dimensional Gaussian function. The experimentally observed distribution of hydrophobicity compared with the theoretical distribution reveals differences. The area of high differences indicates the ligation site. Availability http://bioinformatics.cm-uj.krakow.pl/activesite PMID:17597871

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

Unusual Structure of the attB Site of the Site-Specific Recombination System of Lactobacillus delbrueckii Bacteriophage mv4

PubMed Central

Auvray, Frédéric; Coddeville, Michèle; Ordonez, Romy Catoira; Ritzenthaler, Paul

1999-01-01

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3′ end of a tRNASer gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNASer gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model. PMID:10572145

The Role of the β5-α11 Loop in the Active-Site Dynamics of Acylated Penicillin-Binding Protein A from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constantsmore » for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5–α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5–α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.« less

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

A deep learning framework for modeling structural features of RNA-binding protein targets

PubMed Central

Zhang, Sai; Zhou, Jingtian; Hu, Hailin; Gong, Haipeng; Chen, Ligong; Cheng, Chao; Zeng, Jianyang

2016-01-01

RNA-binding proteins (RBPs) play important roles in the post-transcriptional control of RNAs. Identifying RBP binding sites and characterizing RBP binding preferences are key steps toward understanding the basic mechanisms of the post-transcriptional gene regulation. Though numerous computational methods have been developed for modeling RBP binding preferences, discovering a complete structural representation of the RBP targets by integrating their available structural features in all three dimensions is still a challenging task. In this paper, we develop a general and flexible deep learning framework for modeling structural binding preferences and predicting binding sites of RBPs, which takes (predicted) RNA tertiary structural information into account for the first time. Our framework constructs a unified representation that characterizes the structural specificities of RBP targets in all three dimensions, which can be further used to predict novel candidate binding sites and discover potential binding motifs. Through testing on the real CLIP-seq datasets, we have demonstrated that our deep learning framework can automatically extract effective hidden structural features from the encoded raw sequence and structural profiles, and predict accurate RBP binding sites. In addition, we have conducted the first study to show that integrating the additional RNA tertiary structural features can improve the model performance in predicting RBP binding sites, especially for the polypyrimidine tract-binding protein (PTB), which also provides a new evidence to support the view that RBPs may own specific tertiary structural binding preferences. In particular, the tests on the internal ribosome entry site (IRES) segments yield satisfiable results with experimental support from the literature and further demonstrate the necessity of incorporating RNA tertiary structural information into the prediction model. The source code of our approach can be found in https://github.com/thucombio/deepnet-rbp. PMID:26467480

Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key.

PubMed

Haupt, V Joachim; Daminelli, Simone; Schroeder, Michael

2013-01-01

Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) - a drug's ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor influence.

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE PAGES

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

iSS-PseDNC: identifying splicing sites using pseudo dinucleotide composition.

PubMed

Chen, Wei; Feng, Peng-Mian; Lin, Hao; Chou, Kuo-Chen

2014-01-01

In eukaryotic genes, exons are generally interrupted by introns. Accurately removing introns and joining exons together are essential processes in eukaryotic gene expression. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapid and effective detection of splice sites that play important roles in gene structure annotation and even in RNA splicing. Although a series of computational methods were proposed for splice site identification, most of them neglected the intrinsic local structural properties. In the present study, a predictor called "iSS-PseDNC" was developed for identifying splice sites. In the new predictor, the sequences were formulated by a novel feature-vector called "pseudo dinucleotide composition" (PseDNC) into which six DNA local structural properties were incorporated. It was observed by the rigorous cross-validation tests on two benchmark datasets that the overall success rates achieved by iSS-PseDNC in identifying splice donor site and splice acceptor site were 85.45% and 87.73%, respectively. It is anticipated that iSS-PseDNC may become a useful tool for identifying splice sites and that the six DNA local structural properties described in this paper may provide novel insights for in-depth investigations into the mechanism of RNA splicing.

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

PubMed

Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

Students investigate environmental restoration site in New Mexico

NASA Astrophysics Data System (ADS)

Ferguson, John F.; Baldridge, W. Scott; Jiracek, George R.; Gonzalez, Victor; Pope, Paul A.

Investigations conducted during the 1997 Summer of Applied Geophysical Experience (SAGE) field course at one site at Los Alamos National Laboratory (LANL) successfully delineated a waste disposal trench dug in the 1940s. The survey, which was popular with the students, provided them with important experience in “real world” geophysical problems and demonstrated that students can obtain useful and important results during a short field exercise.The utility of the magnetic, seismic refraction, and ground-penetrating radar methods will be demonstrated on a profile through one of the major waste trenches at the site. The magnetic and radar methods are sensitive to the presence of metallic objects buried within the trenches. A low-velocity trench structure is defined by the seismic refraction data. Models of the trench structure are both accurate (linear dimensions are probably good to within a few meters) and somewhat different from prior expectations.

Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

DOE PAGES

Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

2015-09-21

The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less

Enzyme Active Site Interactions by Raman/FTIR, NMR, and Ab Initio Calculations

PubMed Central

Deng, Hua

2017-01-01

Characterization of enzyme active site structure and interactions at high resolution is important for the understanding of the enzyme catalysis. Vibrational frequency and NMR chemical shift measurements of enzyme-bound ligands are often used for such purpose when X-ray structures are not available or when higher resolution active site structures are desired. This review is focused on how ab initio calculations may be integrated with vibrational and NMR chemical shift measurements to quantitatively determine high-resolution ligand structures (up to 0.001 Å for bond length and 0.01 Å for hydrogen bonding distance) and how interaction energies between bound ligand and its surroundings at the active site may be determined. Quantitative characterization of substrate ionic states, bond polarizations, tautomeric forms, conformational changes and its interactions with surroundings in enzyme complexes that mimic ground state or transition state can provide snapshots for visualizing the substrate structural evolution along enzyme-catalyzed reaction pathway. Our results have shown that the integration of spectroscopic studies with theoretical computation greatly enhances our ability to interpret experimental data and significantly increases the reliability of the theoretical analysis. PMID:24018325

Metal Ion Dependence of the Matrix Metalloproteinase-1 Mechanism.

PubMed

Yang, Hao; Makaroff, Katherine; Paz, Nicholas; Aitha, Mahesh; Crowder, Michael W; Tierney, David L

2015-06-16

Matrix metalloproteinase-1 (MMP-1) plays crucial roles in disease-related physiologies and pathological processes in the human body. We report here solution studies of MMP-1, including characterization of a series of mutants designed to bind metal in either the catalytic site or the structural site (but not both). Circular dichroism and fluorescence spectroscopy of the mutants demonstrate the importance of the structural Zn(II) in maintaining both secondary and tertiary structure, while UV-visible, nuclear magnetic resonance, electron paramagnetic resonance, and extended X-ray absorption fine structure show its presence influences the catalytic metal ion's coordination number. The mutants allow us to demonstrate convincingly the preparation of a mixed-metal analogue, Co(C)Zn(S)-MMP-1, with Zn(II) in the structural site and Co(II) in the catalytic site. Stopped-flow fluorescence of the native form, Zn(C)Zn(S)-MMP-1, and the mixed-metal Co(C)Zn(S)-MMP-1 analogue shows that the internal fluorescence of a nearby Trp residue is modulated with catalysis and can be used to monitor reactivity under a number of conditions, opening the door to substrate profiling.

Evaluation of genetic population structure of smallmouth bass in the Susquehanna River basin, Pennsylvania

USGS Publications Warehouse

Schall, Megan K.; Bartron, Meredith L.; Wertz, Timothy; Niles, Jonathan M.; Shaw, Cassidy H.; Wagner, Tyler

2017-01-01

The Smallmouth Bass Micropterus dolomieu was introduced into the Susquehanna River basin, Pennsylvania, nearly 150 years ago. Since introduction, it has become an economically and ecologically important species that supports popular recreational fisheries. It is also one of the most abundant top predators in the system. Currently, there is no information on the level of genetic diversity or genetic structuring that may have occurred since introduction. An understanding of genetic diversity is important for the delineation of management units and investigation of gene flow at various management scales. The goals of this research were to investigate population genetic structure of Smallmouth Bass at sites within the Susquehanna River basin and to assess genetic differentiation relative to Smallmouth Bass at an out-of-basin site (Allegheny River, Pennsylvania) located within the species’ native range. During spring 2015, fin clips (n = 1,034) were collected from adults at 11 river sites and 13 tributary sites in the Susquehanna River basin and at one site on the Allegheny River. Fin clips were genotyped at 12 polymorphic microsatellite loci. Based on our results, adults sampled throughout the Susquehanna River basin did not represent separate genetic populations. There were only subtle differences in genetic diversity among sites (mean pairwise genetic differentiation index FST = 0.012), and there was an overall lack of population differentiation (K = 3 admixed populations). The greatest genetic differentiation was observed between fish collected from the out-of-basin site and those from the Susquehanna River basin sites. Knowledge that separate genetic populations of Smallmouth Bass do not exist in the Susquehanna River basin is valuable information for fisheries management in addition to providing baseline genetic data on an introduced sport fish population.

Diversity and structure of Chironomidae communities in relation to water quality differences in the Swartkops River

NASA Astrophysics Data System (ADS)

Odume, O. N.; Muller, W. J.

The Swartkops River is an important freshwater ecosystem in South Africa. But owing to its location, it suffers varying degrees of human induced impacts which include industrial and domestic effluent discharges, deforestation as well as agricultural land use which have negatively impacted on the water quality. Diversity and community composition of aquatic insects are frequently used to assess environmental water quality status. Chironomids occupy extremely varied biotopes. Their extraordinary ecological range and environmental sensitivity make them particularly useful for assessing and interpreting changes in water quality of aquatic ecosystems. The community structure of chironomid larvae was investigated at four sites in the Swartkops River and effects of different chemical and physical variables on their distribution were explored. Chironomid larvae were collected using the South African Scoring System version 5 (SASS5) protocol. A total of 26 taxa from four sampling sites in the Swartkops River were identified. Margalef’s species richness index, equitability, Shannon and Simpson diversity indices were highest at site 1 (reference site). The downstream sites contained 6-20 taxa compared to the 25 taxa at site 1. Site 1 was characterised by the subfamilies Orthocladiinae, Tanypodinae and the tribe Tanytarsini while the impacted sites were characterised by Orthocladiinae and Chironomini. Chironomus spp., Dirotendipes sp., Kiefferulus sp. and Tanypus sp. seemed to be tolerant to pollution, occurring in high abundance at sites 2, 3 and 4. In contrast, Polypedilum sp., Tanytarsus sp., Orthocladius sp., Cricotopus spp. and Ablabesmyia sp. appeared to be more sensitive taxa, being less common at the impacted sites (sites 2, 3 and 4). Five days biochemical oxygen demand, dissolved oxygen, electrical conductivity, orthophosphate-phosphorus and total inorganic nitrogen were among the important variables that determine the observed chironomid community structure. Results revealed that the family Chironomidae, identified to species or genus, can be use to assess environmental water quality status in South African freshwater ecosystems.

Human deoxyhaemoglobin-2,3-diphosphoglycerate complex low-salt structure at 2.5 A resolution.

PubMed

Richard, V; Dodson, G G; Mauguen, Y

1993-09-20

The haemoglobin-2,3-diphosphoglycerate complex structure has been solved at 2.5 A resolution using crystals grown from low-salt solutions. The results show some important differences with the precedent haemoglobin-2,3-diphosphoglycerate high-salt structure solved by Arnone. First, we observe a loss of symmetry in the binding site, secondly both of the lysine residues 82 beta interact with 2,3-diphosphoglycerate at the same time, each making two contacts. This level of interaction is in agreement with the functional behaviour of natural haemoglobin mutants with mutations at the 2,3-diphosphoglycerate binding site.

Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

NASA Astrophysics Data System (ADS)

Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

2015-01-01

Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

The crystal structure of the AgamOBP1•Icaridin complex reveals alternative binding modes and stereo-selective repellent recognition.

PubMed

Drakou, Christina E; Tsitsanou, Katerina E; Potamitis, Constantinos; Fessas, Dimitrios; Zervou, Maria; Zographos, Spyros E

2017-01-01

Anopheles gambiae Odorant Binding Protein 1 in complex with the most widely used insect repellent DEET, was the first reported crystal structure of an olfactory macromolecule with a repellent, and paved the way for OBP1-structure-based approaches for discovery of new host-seeking disruptors. In this work, we performed STD-NMR experiments to directly monitor and verify the formation of a complex between AgamOBP1 and Icaridin, an efficient DEET alternative. Furthermore, Isothermal Titration Calorimetry experiments provided evidence for two Icaridin-binding sites with different affinities (Kd = 0.034 and 0.714 mM) and thermodynamic profiles of ligand binding. To elucidate the binding mode of Icaridin, the crystal structure of AgamOBP1•Icaridin complex was determined at 1.75 Å resolution. We found that Icaridin binds to the DEET-binding site in two distinct orientations and also to a novel binding site located at the C-terminal region. Importantly, only the most active 1R,2S-isomer of Icaridin's equimolar diastereoisomeric mixture binds to the AgamOBP1 crystal, providing structural evidence for the possible contribution of OBP1 to the stereoselectivity of Icaridin perception in mosquitoes. Structural analysis revealed two ensembles of conformations differing mainly in spatial arrangement of their sec-butyl moieties. Moreover, structural comparison with DEET indicates a common recognition mechanism for these structurally related repellents. Ligand interactions with both sites and binding modes were further confirmed by 2D 1 H- 15 N HSQC NMR spectroscopy. The identification of a novel repellent-binding site in AgamOBP1 and the observed structural conservation and stereoselectivity of its DEET/Icaridin-binding sites open new perspectives for the OBP1-structure-based discovery of next-generation insect repellents.

The oligomeric architecture of the archaeal exosome is important for processive and efficient RNA degradation.

PubMed

Audin, Maxime J C; Wurm, Jan Philip; Cvetkovic, Milos A; Sprangers, Remco

2016-04-07

The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3' end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3.

PubMed

Shen, Hujun; Deng, Mingsen; Zhang, Yachao

2017-10-01

Recent crystal structures of RNA-dependent RNA polymerase (3D pol ) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3D pol in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3D pol . Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

PubMed Central

Jaffe, Eileen K.; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J.; Myler, Peter J.; Roos, David S.

2011-01-01

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors. PMID:21383008

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori.

PubMed

Blum, Faith C; Hu, Heidi Q; Servetas, Stephanie L; Benoit, Stéphane L; Maier, Robert J; Maroney, Michael J; Merrell, D Scott

2017-01-01

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori

PubMed Central

Servetas, Stephanie L.; Benoit, Stéphane L.; Maier, Robert J.; Maroney, Michael J.

2017-01-01

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance. PMID:28809946

The 1978 and 1979 Excavations at Strawberry Island in the McNary Reservoir.

DTIC Science & Technology

1983-01-01

Schalk et. al 9. PERFORMING ORGANIZATION NAME AND ADDRESS Laboratory of Archaeology and History ’..’ashington State university Pullman...137 Synopsis of Site Occupation History 139 Site Structure and Spatial Organization 140 A Model for Late Prehistoric Change in Settlement... organization in large housepit sites on the Plateau,- it was his belief that this subject was one of considerable importance for future archaeology in the

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

PubMed Central

Chouquet, Anne; Païdassi, Helena; Ling, Wai Li; Frachet, Philippe; Houen, Gunnar; Arlaud, Gérard J.; Gaboriaud, Christine

2011-01-01

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism. PMID:21423620

GenProBiS: web server for mapping of sequence variants to protein binding sites.

PubMed

Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

2017-07-03

Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nickel binding to NikA: an additional binding site reconciles spectroscopy, calorimetry and crystallography.

PubMed

Addy, Christine; Ohara, Masato; Kawai, Fumihiro; Kidera, Akinori; Ikeguchi, Mitsunori; Fuchigami, Sotaro; Osawa, Masanori; Shimada, Ichio; Park, Sam-Yong; Tame, Jeremy R H; Heddle, Jonathan G

2007-02-01

Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.

Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan

2015-10-27

The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. Themore » results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.« less

Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

PubMed

Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

2014-07-29

ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

Structural hierarchy as a key to complex phase selection in Al-Sm

NASA Astrophysics Data System (ADS)

Ye, Z.; Zhang, F.; Sun, Y.; Nguyen, M. C.; Zhou, S. H.; Zhou, L.; Meng, F.; Ott, R. T.; Park, E.; Besser, M. F.; Kramer, M. J.; Ding, Z. J.; Mendelev, M. I.; Wang, C. Z.; Napolitano, R. E.; Ho, K. M.

2017-10-01

Investigating the unknown structure of the complex cubic phase, previously observed to crystallize from melt-spun amorphous Al-10 at.% Sm alloy, we determine the structure in full site-occupancy detail, highlighting several critical structural features that govern the far-from-equilibrium phase selection pathway. Using an efficient genetic algorithm combining molecular dynamics, density functional theory, and x-ray diffraction, the structure is clearly identified as body-centered cubic I m 3 ¯m (No. 229) with ˜140 atoms per cubic unit cell and a lattice parameter of 1.4 nm. The complex structure is further refined to elucidate the detailed site occupancy, revealing full Sm occupancy on 6b sites and split Sm/Al occupancy on 16f sites. Based on the refined site occupancy associated with the experimentally observed phase, we term this phase ɛ -A l60S m11 (bcc), corresponding to the limiting situation when all 16f sites are occupied by Sm. However, it should be recognized that the range of solubility enabled by split occupancy at Sm sites is an important feature in phase selection under experimental conditions, permitting an avenue for transition with little or no chemical partitioning. Our analysis shows that the ɛ -A l60S m11 (bcc) exhibits a "3-6-6-1" first-shell packing around Sm centers on 16f sites, the same dominant motif exhibited by the undercooled liquid. The coincident motif supports the notion that liquid/glass ordering at high undercooling may give rise to topological invariants between the noncrystalline and crystalline states that provide kinetic pathways to metastable phases that are not accessible during near-equilibrium processing.

Structural hierarchy as a key to complex phase selection in Al-Sm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Z.; Zhang, F.; Sun, Y.

Investigating the unknown structure of the complex cubic phase, previously observed to crystallize from melt-spun amorphous Al–10 at.% Sm alloy, we determine the structure in full site-occupancy detail, highlighting several critical structural features that govern the far-from-equilibrium phase selection pathway. Using an efficient genetic algorithm combining molecular dynamics, density functional theory, and x-ray diffraction, the structure is clearly identified as body-centered cubic Im¯3m (No. 229) with ~140 atoms per cubic unit cell and a lattice parameter of 1.4 nm. The complex structure is further refined to elucidate the detailed site occupancy, revealing full Sm occupancy on 6b sites and splitmore » Sm/Al occupancy on 16f sites. Based on the refined site occupancy associated with the experimentally observed phase, we term this phase ε–Al 60Sm 11(bcc), corresponding to the limiting situation when all 16f sites are occupied by Sm. However, it should be recognized that the range of solubility enabled by split occupancy at Sm sites is an important feature in phase selection under experimental conditions, permitting an avenue for transition with little or no chemical partitioning. Our analysis shows that the ε–Al 60Sm 11(bcc) exhibits a “3-6-6-1” first-shell packing around Sm centers on 16f sites, the same dominant motif exhibited by the undercooled liquid. Here, the coincident motif supports the notion that liquid/glass ordering at high undercooling may give rise to topological invariants between the noncrystalline and crystalline states that provide kinetic pathways to metastable phases that are not accessible during near-equilibrium processing.« less

Structural hierarchy as a key to complex phase selection in Al-Sm

DOE PAGES

Ye, Z.; Zhang, F.; Sun, Y.; ...

2017-10-12

Investigating the unknown structure of the complex cubic phase, previously observed to crystallize from melt-spun amorphous Al–10 at.% Sm alloy, we determine the structure in full site-occupancy detail, highlighting several critical structural features that govern the far-from-equilibrium phase selection pathway. Using an efficient genetic algorithm combining molecular dynamics, density functional theory, and x-ray diffraction, the structure is clearly identified as body-centered cubic Im¯3m (No. 229) with ~140 atoms per cubic unit cell and a lattice parameter of 1.4 nm. The complex structure is further refined to elucidate the detailed site occupancy, revealing full Sm occupancy on 6b sites and splitmore » Sm/Al occupancy on 16f sites. Based on the refined site occupancy associated with the experimentally observed phase, we term this phase ε–Al 60Sm 11(bcc), corresponding to the limiting situation when all 16f sites are occupied by Sm. However, it should be recognized that the range of solubility enabled by split occupancy at Sm sites is an important feature in phase selection under experimental conditions, permitting an avenue for transition with little or no chemical partitioning. Our analysis shows that the ε–Al 60Sm 11(bcc) exhibits a “3-6-6-1” first-shell packing around Sm centers on 16f sites, the same dominant motif exhibited by the undercooled liquid. Here, the coincident motif supports the notion that liquid/glass ordering at high undercooling may give rise to topological invariants between the noncrystalline and crystalline states that provide kinetic pathways to metastable phases that are not accessible during near-equilibrium processing.« less

Structures for the ABO(H) Blood Group: Which Textbook Is Correct?

NASA Astrophysics Data System (ADS)

Risley, John M.

2007-09-01

Six textbooks and two Internet sites show different structures for the A, B, and O(H) antigens of the ABO(H) blood group. However, none of the structures identified as the A, B, and O(H) antigens are correct. The O(H) antigen is a disaccharide, on which the trisaccharide A and B antigens are synthesized. The structures shown in the textbooks and at the Internet sites contain the O(H), A, and B antigens attached at the nonreducing end of various heterosaccharide cores of glycoproteins and glycolipids that are not a part of the specific blood group. This article emphasizes the correct molecular structures because it is important to distinguish between those carbohydrates that make up the antigens and those that are not part of the antigenic structures.

Structural determinants of ubiquitin-CXC chemokine receptor 4 interaction.

PubMed

Saini, Vikas; Marchese, Adriano; Tang, Wei-Jen; Majetschak, Matthias

2011-12-23

Ubiquitin, a post-translational protein modifier inside the cell, functions as a CXC chemokine receptor (CXCR) 4 agonist outside the cell. However, the structural determinants of the interaction between extracellular ubiquitin and CXCR4 remain unknown. Utilizing C-terminal truncated ubiquitin and ubiquitin mutants, in which surface residues that are known to interact with ubiquitin binding domains in interacting proteins are mutated (Phe-4, Leu-8, Ile-44, Asp-58, Val-70), we provide evidence that the ubiquitin-CXCR4 interaction follows a two-site binding mechanism in which the hydrophobic surfaces surrounding Phe-4 and Val-70 are important for receptor binding, whereas the flexible C terminus facilitates receptor activation. Based on these findings and the available crystal structures, we then modeled the ubiquitin-CXCR4 interface with the RosettaDock software followed by small manual adjustments, which were guided by charge complementarity and anticipation of a conformational switch of CXCR4 upon activation. This model suggests three residues of CXCR4 (Phe-29, Phe-189, Lys-271) as potential interaction sites. Binding studies with HEK293 cells overexpressing wild type and CXCR4 after site-directed mutagenesis confirm that these residues are important for ubiquitin binding but that they do not contribute to the binding of stromal cell-derived factor 1α. Our findings suggest that the structural determinants of the CXCR4 agonist activity of ubiquitin mimic the typical structure-function relationship of chemokines. Furthermore, we provide evidence for separate and specific ligand binding sites on CXCR4. As exogenous ubiquitin has been shown to possess therapeutic potential, our findings are expected to facilitate the structure-based design of new compounds with ubiquitin-mimetic actions on CXCR4.

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy.

PubMed

Lawrenz, Morgan; Wereszczynski, Jeff; Amaro, Rommie; Walker, Ross; Roitberg, Adrian; McCammon, J Andrew

2010-08-15

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium-bound and calcium-free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can "clamp" the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure-based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. 2010 Wiley-Liss, Inc.

Determination of structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

PubMed Central

Wu, Wei; Park, Kyung-Tae; Holyoak, Todd; Lutkenhaus, Joe

2011-01-01

Summary The three Min proteins spatially regulate Z ring positioning in E. coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP-dependency of MinC and MinE binding to MinD. PMID:21231967

Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs{sup +}-selective binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo

2015-03-01

The tertiary structure of a β-lactamase derived from the halobacterium Chromohalobacter sp. 560 (HaBLA) was determined by X-ray crystallography. Three unique Sr{sup 2+}-binding sites and one Cs{sup +}-binding site were discovered in the HaBLA molecule. Environmentally friendly absorbents are needed for Sr{sup 2+} and Cs{sup +}, as the removal of the radioactive Sr{sup 2+} and Cs{sup +} that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs{sup +}more » or Sr{sup 2+}. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P3{sub 1} using X-ray crystallography. Moreover, the locations of bound Sr{sup 2+} and Cs{sup +} ions were identified by anomalous X-ray diffraction. The location of one Cs{sup +}-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na{sup +} (90 mM Na{sup +}/10 mM Cs{sup +}). From an activity assay using isothermal titration calorimetry, the bound Sr{sup 2+} and Cs{sup +} ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs{sup +}-binding site provides important information that is useful for the design of artificial Cs{sup +}-binding sites that may be useful in the bioremediation of radioactive isotopes.« less

Changes in assembly processes in soil bacterial communities following a wildfire disturbance.

PubMed

Ferrenberg, Scott; O'Neill, Sean P; Knelman, Joseph E; Todd, Bryan; Duggan, Sam; Bradley, Daniel; Robinson, Taylor; Schmidt, Steven K; Townsend, Alan R; Williams, Mark W; Cleveland, Cory C; Melbourne, Brett A; Jiang, Lin; Nemergut, Diana R

2013-06-01

Although recent work has shown that both deterministic and stochastic processes are important in structuring microbial communities, the factors that affect the relative contributions of niche and neutral processes are poorly understood. The macrobiological literature indicates that ecological disturbances can influence assembly processes. Thus, we sampled bacterial communities at 4 and 16 weeks following a wildfire and used null deviation analysis to examine the role that time since disturbance has in community assembly. Fire dramatically altered bacterial community structure and diversity as well as soil chemistry for both time-points. Community structure shifted between 4 and 16 weeks for both burned and unburned communities. Community assembly in burned sites 4 weeks after fire was significantly more stochastic than in unburned sites. After 16 weeks, however, burned communities were significantly less stochastic than unburned communities. Thus, we propose a three-phase model featuring shifts in the relative importance of niche and neutral processes as a function of time since disturbance. Because neutral processes are characterized by a decoupling between environmental parameters and community structure, we hypothesize that a better understanding of community assembly may be important in determining where and when detailed studies of community composition are valuable for predicting ecosystem function.

Changes in assembly processes in soil bacterial communities following a wildfire disturbance

PubMed Central

Ferrenberg, Scott; O'Neill, Sean P; Knelman, Joseph E; Todd, Bryan; Duggan, Sam; Bradley, Daniel; Robinson, Taylor; Schmidt, Steven K; Townsend, Alan R; Williams, Mark W; Cleveland, Cory C; Melbourne, Brett A; Jiang, Lin; Nemergut, Diana R

2013-01-01

Although recent work has shown that both deterministic and stochastic processes are important in structuring microbial communities, the factors that affect the relative contributions of niche and neutral processes are poorly understood. The macrobiological literature indicates that ecological disturbances can influence assembly processes. Thus, we sampled bacterial communities at 4 and 16 weeks following a wildfire and used null deviation analysis to examine the role that time since disturbance has in community assembly. Fire dramatically altered bacterial community structure and diversity as well as soil chemistry for both time-points. Community structure shifted between 4 and 16 weeks for both burned and unburned communities. Community assembly in burned sites 4 weeks after fire was significantly more stochastic than in unburned sites. After 16 weeks, however, burned communities were significantly less stochastic than unburned communities. Thus, we propose a three-phase model featuring shifts in the relative importance of niche and neutral processes as a function of time since disturbance. Because neutral processes are characterized by a decoupling between environmental parameters and community structure, we hypothesize that a better understanding of community assembly may be important in determining where and when detailed studies of community composition are valuable for predicting ecosystem function. PMID:23407312

Structural insight to mutation effects uncover a common allosteric site in class C GPCRs.

PubMed

Harpsøe, Kasper; Boesgaard, Michael W; Munk, Christian; Bräuner-Osborne, Hans; Gloriam, David E

2017-04-15

Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry. Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. We uncover one common site for both positive and negative modulators with different amino acid layouts that can be utilized to obtain selectivity. Additionally, we show a large potential for structure-based modulator design, especially for four orphan receptors with high similarity to the crystal structures. All collated mutagenesis data is available in the GPCRdb mutation browser at http://gpcrdb.org/mutations/ and can be analyzed online or downloaded in excel format. david.gloriam@sund.ku.dk. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Modeled structure of trypanothione reductase of Leishmania infantum.

PubMed

Singh, Bishal K; Sarkar, Nandini; Jagannadham, M V; Dubey, Vikash K

2008-06-30

Trypanothione reductase is an important target enzyme for structure-based drug design against Leishmania. We used homology modeling to construct a three-dimensional structure of the trypanothione reductase (TR) of Leishmania infantum. The structure shows acceptable Ramachandran statistics and a remarkably different active site from glutathione reductase(GR). Thus, a specific inhibitor against TR can be designed without interfering with host (human) GR activity.

Design of Single-Site Photocatalyst using Metal-Organic Framework as Matrix.

PubMed

Wen, Meicheng; Mori, Kohsuke; Kuwahara, Yasutaka; An, Taicheng; Yamashita, Hiromi

2018-05-14

Single-site photocatalyst generally displays excellent photocatalytic activtiy and considerable high stability as compared to homogeneous catalytic system. A rational structural design of single-site photocatalyst with isolated, uniform and spatially separated active sites in a given solid is of prime importance to achieve high photocatalytic activity. Intense attentions have been focused on the engineering and fabrication of single-site photocatalys by using porous materials as platform. Metal-organic frameworks (MOFs) hold great potential for the design and fabrication of single-site photocatalysts due to their remarkable porosity, ultrahigh surface area, extraordinary tailorability and significant diversity. MOFs can provide abundant number of binding sites for anchoring active sites, result in significant enhancement of photocatalytic performance. In this focus review, the development of single-site MOF photocatalysts that perform in important and challenging chemical redox reaction such as photocatalytic water splitting, photocatalytic CO₂ conversion and organic transformations is summarized thoroughly. The successful strategies applied for the construction of single-site MOF photocatalysts and major challenge toward practical application was summarized and pointed out, respectively. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Habitat structure mediates predation risk for sedentary prey: Experimental tests of alternative hypotheses

USGS Publications Warehouse

Chalfoun, A.D.; Martin, T.E.

2009-01-01

Predation is an important and ubiquitous selective force that can shape habitat preferences of prey species, but tests of alternative mechanistic hypotheses of habitat influences on predation risk are lacking. 2. We studied predation risk at nest sites of a passerine bird and tested two hypotheses based on theories of predator foraging behaviour. The total-foliage hypothesis predicts that predation will decline in areas of greater overall vegetation density by impeding cues for detection by predators. The potential-prey-site hypothesis predicts that predation decreases where predators must search more unoccupied potential nest sites. 3. Both observational data and results from a habitat manipulation provided clear support for the potential-prey-site hypothesis and rejection of the total-foliage hypothesis. Birds chose nest patches containing both greater total foliage and potential nest site density (which were correlated in their abundance) than at random sites, yet only potential nest site density significantly influenced nest predation risk. 4. Our results therefore provided a clear and rare example of adaptive nest site selection that would have been missed had structural complexity or total vegetation density been considered alone. 5. Our results also demonstrated that interactions between predator foraging success and habitat structure can be more complex than simple impedance or occlusion by vegetation. ?? 2008 British Ecological Society.

Molecular dynamics simulation studies of novel β-lactamase inhibitor.

PubMed

Ul Haq, Farhan; Abro, Asma; Raza, Saad; Liedl, Klaus R; Azam, Syed Sikander

2017-06-01

New Delhi Metallo-β-Lactamase-1 (NDM-1) has drawn great attention due to its diverse antibiotic resistant activity. It can hydrolyze almost all clinically available β-lactam antibiotics. To inhibit the activity of NDM-1 a new strategy is proposed using computational methods. Molecular dynamics (MD) simulations are used to analyze the molecular interactions between selected inhibitor candidates and NDM-1 structure. The enzyme-ligand complex is subject to binding free energy calculations using MM(PB/GB)SA methods. The role of each residue of the active site contributing in ligand binding affinity is explored using energy decomposition analysis. Furthermore, a hydrogen bonding network between ligand and enzyme active site is observed and key residues are identified ensuring that the ligand stays inside the active site and maintains its movement towards the active site pocket. A production run of 150ns is carried out and results are analyzed using root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) to explain the stability of enzyme ligand complex. Important active site residue e.g. PHE70, VAL73, TRP93, HIS122, GLN123, ASP124, HIS189, LYS216, CYS208, LYS211, ALA215, HIS250, and SER251 were observed to be involved in ligand attachemet inside the active site pocket, hence depicting its inhibitor potential. Hydrogen bonds involved in structural stability are analyzed through radial distribution function (RDF) and contribution of important residues involved in ligand movement is explained using a novel analytical tool, axial frequency distribution (AFD) to observe the role of important hydrogen bonding partners between ligand atoms and active site residues. Copyright © 2017 Elsevier Inc. All rights reserved.

Influence of gamma-ray skyshine on nuclear facilities design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohta, M.; Tsuji, M.; Kimura, Y.

1986-01-01

In safety analysis of nuclear facilities, skyshine dose rate at site boundary is one of the most important shielding design problems. For nuclear power stations in Japan, the skyshine dose rate at the site boundary has been specified not to exceed 5 mR/yr by the authorities, including total dose contribution from all structures on site, and this guide is commonly applied to other nuclear fuel cycle facilities. Therefore the design criterion dose of each structure on site is, considering plot planning, shielding condition, and so on, defined as a value <5 mR/yr. The purpose of this study is to investigatemore » how skyshine dose standards or other factors have an influence on the design of nuclear facilities, in a parametric survey of gamma-ray skyshine.« less

Microtremors for seismic response assessments of important modern and historical structures of Crete

NASA Astrophysics Data System (ADS)

Margarita, Moisidi; Filippos, Vallianatos

2017-12-01

Strengthening seismic risk resilience undertaken by the civil protection authorities is an important issue towards to the guidelines given by Sendai Framework, 2013 European Union Civil Protection legislation and the global agenda 2030 for sustainable development. Moreover, in recent years it has been emphasized that site effect specifications are important issues for the seismic hazard assessments of modern, historical and monumental structures. This study assess the frequencies of vibration of historical, monumental and modern structures in the cities of Chania, Rethymno and Heraklion of Crete using ambient noise recordings processed through the Horizontal to Vertical spectral ratio and examines potential soil-structure interaction phenomena. Examples of the seismic response of high rise structures such as a church bell tower and the lighthouses in Chania are presented.

Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Aimee; Higgins, Darren E.; Panne, Daniel

2009-07-22

The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity.more » The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.« less

Water molecules in the nucleotide binding cleft of actin: effects on subunit conformation and implications for ATP hydrolysis.

PubMed

Saunders, Marissa G; Voth, Gregory A

2011-10-14

In the monomeric actin crystal structure, the positions of a highly organized network of waters are clearly visible within the active site. However, the recently proposed models of filamentous actin (F-actin) did not extend to including these waters. Since the water network is important for ATP hydrolysis, information about water position is critical to understanding the increased rate of catalysis upon filament formation. Here, we show that waters in the active site are essential for intersubdomain rotational flexibility and that they organize the active-site structure. Including the crystal structure waters during simulation setup allows us to observe distinct changes in the active-site structure upon the flattening of the actin subunit, as proposed in the Oda model for F-actin. We identify changes in both protein position and water position relative to the phosphate tail that suggest a mechanism for accelerating the rate of nucleotide hydrolysis in F-actin by stabilizing charge on the β-phosphate and by facilitating deprotonation of catalytic water. Copyright © 2011 Elsevier Ltd. All rights reserved.

Crystallographic snapshots of active site metal shift in E. coli fructose 1,6-bisphosphate aldolase.

PubMed

Tran, Huyen-Thi; Lee, Seon-Hwa; Ho, Thien-Hoang; Hong, Seung-Hye; Huynh, Kim-Hung; Ahn, Yeh-Jin; Oh, Deok-Kun; Kang, Lin-Woo

2016-12-01

Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 Å and 2.0 Å. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2＋ ion. Citrate and TRIS bound EcFBA structures showed Zn2＋ position exclusively at Zn2. Crystallographic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2＋ within EcFBA without losing it. [BMB Reports 2016; 49(12): 681-686].

UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

PubMed

Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

2016-01-04

The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Free and ATP-bound structures of Ap4A hydrolase from Aquifex aeolicus V5.

PubMed

Jeyakanthan, Jeyaraman; Kanaujia, Shankar Prasad; Nishida, Yuya; Nakagawa, Noriko; Praveen, Surendran; Shinkai, Akeo; Kuramitsu, Seiki; Yokoyama, Shigeyuki; Sekar, Kanagaraj

2010-02-01

Asymmetric diadenosine tetraphosphate (Ap(4)A) hydrolases degrade the metabolite Ap(4)A back into ATP and AMP. The three-dimensional crystal structure of Ap(4)A hydrolase (16 kDa) from Aquifex aeolicus has been determined in free and ATP-bound forms at 1.8 and 1.95 A resolution, respectively. The overall three-dimensional crystal structure of the enzyme shows an alphabetaalpha-sandwich architecture with a characteristic loop adjacent to the catalytic site of the protein molecule. The ATP molecule is bound in the primary active site and the adenine moiety of the nucleotide binds in a ring-stacking arrangement equivalent to that observed in the X-ray structure of Ap(4)A hydrolase from Caenorhabditis elegans. Binding of ATP in the active site induces local conformational changes which may have important implications in the mechanism of substrate recognition in this class of enzymes. Furthermore, two invariant water molecules have been identified and their possible structural and/or functional roles are discussed. In addition, modelling of the substrate molecule at the primary active site of the enzyme suggests a possible path for entry and/or exit of the substrate and/or product molecule.

Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nuemket, Nipawan; Tanaka, Yoshikazu; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810

2011-07-29

Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinummore » neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.« less

Structure of Thermotoga maritima Stationary Phase Survival Protein SurE: A Novel Acid Phosphatase

PubMed Central

Zhang, R.-G.; Skarina, T.; Katz, J.E.; Beasley, S.; Khachatryan, A.; Vyas, S.; Arrowsmith, C.H.; Clarke, S.; Edwards, A.; Joachimiak, A.; Savchenko, A.

2009-01-01

Summary Background The rpoS, nlpD, pcm, and surE genes are among many whose expression is induced during the stationary phase of bacterial growth. rpoS codes for the stationary-phase RNA polymerase σ subunit, and nlpD codes for a lipoprotein. The pcm gene product repairs damaged proteins by converting the atypical isoaspartyl residues back to L-aspartyls. The physiological and biochemical functions of surE are unknown, but its importance in stress is supported by the duplication of the surE gene in E. coli subjected to high-temperature growth. The pcm and surE genes are highly conserved in bacteria, archaea, and plants. Results The structure of SurE from Thermotoga maritima was determined at 2.0 Å. The SurE monomer is composed of two domains; a conserved N-terminal domain, a Rossman fold, and a C-terminal oligomerization domain, a new fold. Monomers form a dimer that assembles into a tetramer. Biochemical analysis suggests that SurE is an acid phosphatase, with an optimum pH of 5.5–6.2. The active site was identified in the N-terminal domain through analysis of conserved residues. Structure-based site-directed point mutations abolished phosphatase activity. T. maritima SurE intra- and inter-subunit salt bridges were identified that may explain the SurE thermostability. Conclusions The structure of SurE provided information about the protein’s fold, oligomeric state, and active site. The protein possessed magnesium-dependent acid phosphatase activity, but the physiologically relevant substrate(s) remains to be identified. The importance of three of the assigned active site residues in catalysis was confirmed by site-directed mutagenesis. PMID:11709173

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-based Markov random field model for representing evolutionary constraints on functional sites.

PubMed

Jeong, Chan-Seok; Kim, Dongsup

2016-02-24

Elucidating the cooperative mechanism of interconnected residues is an important component toward understanding the biological function of a protein. Coevolution analysis has been developed to model the coevolutionary information reflecting structural and functional constraints. Recently, several methods have been developed based on a probabilistic graphical model called the Markov random field (MRF), which have led to significant improvements for coevolution analysis; however, thus far, the performance of these models has mainly been assessed by focusing on the aspect of protein structure. In this study, we built an MRF model whose graphical topology is determined by the residue proximity in the protein structure, and derived a novel positional coevolution estimate utilizing the node weight of the MRF model. This structure-based MRF method was evaluated for three data sets, each of which annotates catalytic site, allosteric site, and comprehensively determined functional site information. We demonstrate that the structure-based MRF architecture can encode the evolutionary information associated with biological function. Furthermore, we show that the node weight can more accurately represent positional coevolution information compared to the edge weight. Lastly, we demonstrate that the structure-based MRF model can be reliably built with only a few aligned sequences in linear time. The results show that adoption of a structure-based architecture could be an acceptable approximation for coevolution modeling with efficient computation complexity.

10 CFR Appendix A to Part 100 - Seismic and Geologic Siting Criteria for Nuclear Power Plants

Code of Federal Regulations, 2014 CFR

2014-01-01

.... purpose General Design Criterion 2 of Appendix A to part 50 of this chapter requires that nuclear power plant structures, systems, and components important to safety be designed to withstand the effects of... proposed sites for nuclear power plants and the suitability of the plant design bases established in...

10 CFR Appendix A to Part 100 - Seismic and Geologic Siting Criteria for Nuclear Power Plants

Code of Federal Regulations, 2013 CFR

2013-01-01

.... purpose General Design Criterion 2 of Appendix A to part 50 of this chapter requires that nuclear power plant structures, systems, and components important to safety be designed to withstand the effects of... proposed sites for nuclear power plants and the suitability of the plant design bases established in...

10 CFR Appendix A to Part 100 - Seismic and Geologic Siting Criteria for Nuclear Power Plants

Code of Federal Regulations, 2012 CFR

2012-01-01

.... purpose General Design Criterion 2 of Appendix A to part 50 of this chapter requires that nuclear power plant structures, systems, and components important to safety be designed to withstand the effects of... proposed sites for nuclear power plants and the suitability of the plant design bases established in...

10 CFR Appendix A to Part 100 - Seismic and Geologic Siting Criteria for Nuclear Power Plants

Code of Federal Regulations, 2011 CFR

2011-01-01

.... purpose General Design Criterion 2 of Appendix A to part 50 of this chapter requires that nuclear power plant structures, systems, and components important to safety be designed to withstand the effects of... proposed sites for nuclear power plants and the suitability of the plant design bases established in...

10 CFR Appendix A to Part 100 - Seismic and Geologic Siting Criteria for Nuclear Power Plants

Code of Federal Regulations, 2010 CFR

2010-01-01

.... purpose General Design Criterion 2 of Appendix A to part 50 of this chapter requires that nuclear power plant structures, systems, and components important to safety be designed to withstand the effects of... proposed sites for nuclear power plants and the suitability of the plant design bases established in...

Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry†

PubMed Central

Wang, Liwen; Qin, Yali; Ilchenko, Serguei; Bohon, Jen; Shi, Wuxian; Cho, Michael W.; Takamoto, Keiji; Chance, Mark R.

2010-01-01

Structural characterization of the HIV envelope protein gp120 is very important to provide an understanding of the protein's immunogenicity and it's binding to cell receptors. So far, crystallographic structure determination of gp120 with an intact V3 loop (in the absence of CD4 co-receptor or antibody) has not been achieved. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for co-receptor binding and viral entry to T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated glycosylation occupancy of gp120-OD8; eleven sites from fifteen glycosylation motifs were determined as having high mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information on protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4/ gp120-OD8 crystal structure revealed a flexible V3 loop structure where the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure. PMID:20825246

Geophysical surveys and archaeological insights at Fort Pierre Chouteau, a frontier trading post on the Middle Missouri

NASA Astrophysics Data System (ADS)

Patton, Margaret Maurine

Fort Pierre Chouteau in present day South Dakota was the most important fur trading post of the American Fur Company in the 1830s, serving as a regional hub for the fur trade. The Fort was sold to the U.S. Military in 1855 for use as a base in the Sioux Wars but was abandoned in 1856. Geophysical surveys and previous excavations indicate evidence of both occupations. Geophysics is an important tool for determining the extent of archaeological sites, yet the relationships between geophysical anomalies and excavation features may not be readily evident. Initial geophysical surveys (Kvamme 2007) were completed to determine the extent of the fur trading Fort, and additional surveys in August 2012 used magnetometry and electrical resistance to determine if evidence of military structures exists outside of the Fort. This study examines connections between excavation features and geophysical anomalies in order to better interpret anomalies inside the Fort palisade. The palisade builder's trench, adobe pavement, post holes, and unknown structures are characterized through the analysis of the excavations and anomalies. The location of one of the military structures outside of the palisade is also identified. As many sites have histories of excavations prior to any geophysical surveys, combining the two sets of information is important in order to more fully understand site layout and the archaeological causes of geophysical anomalies.

Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles

NASA Astrophysics Data System (ADS)

Shih, Yu-Ling; Le, Trung; Rothfield, Lawrence

2003-06-01

The MinCDE proteins of Escherichia coli are required for proper placement of the division septum at midcell. The site selection process requires the rapid oscillatory redistribution of the proteins from pole to pole. We report that the three Min proteins are organized into extended membrane-associated coiled structures that wind around the cell between the two poles. The pole-to-pole oscillation of the proteins reflects oscillatory changes in their distribution within the coiled structure. We also report that the E. coli MreB protein, which is required for maintaining the rod shape of the cell, also forms extended coiled structures, which are similar to the MreB structures that have previously been reported in Bacillus subtilis. The MreB and MinCDE coiled arrays do not appear identical. The results suggest that at least two functionally distinct cytoskeletal-like elements are present in E. coli and that structures of this type can undergo dynamic changes that play important roles in division site placement and possibly other aspects of the life of the cell.

Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

PubMed

Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

2016-05-12

Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

DOE Office of Scientific and Technical Information (OSTI.GOV)

J Chang; S Xiang; K Xiang

The 5' {yields} 3' exoribonucleases (XRNs) have important functions in transcription, RNA metabolism and RNA interference. The structure of Rat1 (also known as Xrn2) showed that the two highly conserved regions of XRNs form a single, large domain that defines the active site of the enzyme. Xrn1 has a 510-residue segment after the conserved regions that is required for activity but is absent from Rat1/Xrn2. Here we report the crystal structures of Kluyveromyces lactis Xrn1 (residues 1-1,245, E178Q mutant), alone and in complex with a Mn{sup 2+} ion in the active site. The 510-residue segment contains four domains (D1-D4), locatedmore » far from the active site. Our mutagenesis and biochemical studies show that their functional importance results from their ability to stabilize the conformation of the N-terminal segment of Xrn1. These domains might also constitute a platform that interacts with protein partners of Xrn1.« less

SusG: A Unique Cell-Membrane-Associated [alpha]-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koropatkin, Nicole M.; Smith, Thomas J.

SusG is an {alpha}-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysismore » demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.« less

Structural basis for the regulation of nuclear import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylation of the nuclear localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakada, Ryohei; Hirano, Hidemi; Structural Biology Research Center, Graduate School of Science, Nagoya University

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in every EBV-positive tumor and is essential for the maintenance, replication, and transcription of the EBV genome in the nucleus of host cells. EBNA1 is a serine phosphoprotein, and it has been shown that phosphorylation of S385 in the nuclear localization signal (NLS) of EBNA1 increases the binding affinity to the nuclear import adaptor importin-α1 as well as importin-α5, and stimulates nuclear import of EBNA1. To gain insights into how phosphorylation of the EBNA1 NLS regulates nuclear import, we have determined the crystal structures of two peptide complexes of importin-α1: onemore » with S385-phosphorylated EBNA1 NLS peptide, determined at 2.0 Å resolution, and one with non-phosphorylated EBNA1 NLS peptide, determined at 2.2 Å resolution. The structures show that EBNA1 NLS binds to the major and minor NLS-binding sites of importin-α1, and indicate that the binding affinity of the EBNA1 NLS to the minor NLS-binding site could be enhanced by phosphorylation of S385 through electrostatic interaction between the phosphate group of phospho-S385 and K392 of importin-α1 (corresponding to R395 of importin-α5) on armadillo repeat 8. - Highlights: • Nuclear import of EBNA1 can be regulated by phosphorylation of NLS. • Crystal structures of importin-α1 bound to the NLS peptides of EBNA1 are solved. • Structures provide insights into how phosphorylation can regulate nuclear import.« less

Long-term experimental warming alters nitrogen-cycling communities but site factors remain the primary drivers of community structure in high arctic tundra soils.

PubMed

Walker, Jennifer K M; Egger, Keith N; Henry, Gregory H R

2008-09-01

Arctic air temperatures are expected to rise significantly over the next century. Experimental warming of arctic tundra has been shown to increase plant productivity and cause community shifts and may also alter microbial community structure. Hence, the objective of this study was to determine whether experimental warming caused shifts in soil microbial communities by measuring changes in the frequency, relative abundance and/or richness of nosZ and nifH genotypes. Five sites at a high arctic coastal lowland were subjected to a 13-year warming experiment using open-top chambers (OTCs). Sites differed by dominant plant community, soil parent material and/or moisture regimen. Six soil cores were collected from each of four replicate OTC and ambient plots at each site and subdivided into upper and lower samples. Differences in frequency and relative abundance of terminal restriction fragments were assessed graphically by two-way cluster analysis and tested statistically with permutational multivariate analysis of variance (ANOVA). Genotypic richness was compared using factorial ANOVA. The genotype frequency, relative abundance and genotype richness of both nosZ and nifH communities differed significantly by site, and by OTC treatment and/or depth at some sites. The site that showed the most pronounced treatment effect was a wet sedge meadow, where community structure and genotype richness of both nosZ and nifH were significantly affected by warming. Although warming was an important factor affecting these communities at some sites at this high arctic lowland, overall, site factors were the main determinants of community structure.

Binding Site Configurations Probe the Structure and Dynamics of the Zinc Finger of NEMO (NF-κB Essential Modulator).

PubMed

Godwin, Ryan C; Melvin, Ryan L; Gmeiner, William H; Salsbury, Freddie R

2017-01-31

Zinc-finger proteins are regulators of critical signaling pathways for various cellular functions, including apoptosis and oncogenesis. Here, we investigate how binding site protonation states and zinc coordination influence protein structure, dynamics, and ultimately function, as these pivotal regulatory proteins are increasingly important for protein engineering and therapeutic discovery. To better understand the thermodynamics and dynamics of the zinc finger of NEMO (NF-κB essential modulator), as well as the role of zinc, we present results of 20 μs molecular dynamics trajectories, 5 μs for each of four active site configurations. Consistent with experimental evidence, the zinc ion is essential for mechanical stabilization of the functional, folded conformation. Hydrogen bond motifs are unique for deprotonated configurations yet overlap in protonated cases. Correlated motions and principal component analysis corroborate the similarity of the protonated configurations and highlight unique relationships of the zinc-bound configuration. We hypothesize a potential mechanism for zinc binding from results of the thiol configurations. The deprotonated, zinc-bound configuration alone predominantly maintains its tertiary structure throughout all 5 μs and alludes rare conformations potentially important for (im)proper zinc-finger-related protein-protein or protein-DNA interactions.

Evaluating Seismic Site Effects at Cultural Heritage Sites in the Mediterranean Area

NASA Astrophysics Data System (ADS)

Imposa, S.; D'Amico, S.; Panzera, F.; Lombardo, G.; Grassi, S.; Betti, M.; Muscat, R.

2017-12-01

Present study concern integrated geophysical and numerical simulation aiming at evaluate the seismic vulnerability of cultural heritage sites. Non-invasive analysis targeted to characterize local site effects as well as dynamic properties of the structure were performed. Data were collected at several locations in the Maltese Archipelago (central Mediterranean) and in some historical buildings located in Catania (Sicily). In particular, passive seismic techniques and H/V data where used to derive 1D velocity models and amplification functions. The dynamic properties of a building are usually described through its natural frequency and the damping ratio. This latter is important in seismic design since it allows one to evaluate the ability of a structure to dissipate the vibration energy during an earthquake. The fundamental frequency of the investigated structure was obtained using ambient vibrations recorded by two or more sensors monitoring the motion at different locations in the building. Accordingly, the fundamental period of several Maltese Watchtowers and some historical buildings of Catania were obtained by computing the ratio between the amplitudes of the Fourier spectrum of horizontal (longitudinal and transverse) components recorded on the top and on the ground floors. Using ANSYS code, the modal analysis was performed to evaluate the first 50 vibration modes with the aim to check the activation of the modal masses and to assess the seismic vulnerability of the tower. The STRATA code was instead adopted in the Catania heritage buildings using as reference earthquake moderate to strong shocks that struck south-eastern Sicily. In most of the investigated buildings is was not possible to identify a single natural frequency but several oscillation modes. These results appear linked to the structural complexity of the edifices, their irregular plan shape and the presence of adjacent structures. The H/V outside the buildings were used to determine predominant frequencies of the soil and to highlight potential site-to-structure resonance. The achieved findings can represent useful clues for further additional engineering investigations aiming at reducing the seismic risk, highlighting how the structural complexity and the local seismic response play an important role on building damage.

An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu

2015-09-23

Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from themore » thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

Naturally Occurring Structural Isomers in Serum IgA1 O-Glycosylation

PubMed Central

Takahashi, Kazuo; Smith, Archer D.; Poulsen, Knud; Kilian, Mogens; Julian, Bruce A.; Mestecky, Jiri; Novak, Jan; Renfrow, Matthew B.

2013-01-01

IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr225, Thr228, Ser230, Ser232 and Thr236 have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC/MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser230, Thr233 or Thr236 produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability. PMID:22067045

Structures of a bifunctional cell wall hydrolase CwlT containing a novel bacterial lysozyme and an NlpC/P60 DL-endopeptidase.

PubMed

Xu, Qingping; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

2014-01-09

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state). © 2013.

Unusual conformation of the SxN motif in the crystal structure of penicillin-binding protein A from Mycobacterium tuberculosis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fedarovich, Alena; Nicholas, Robert A.; Davies, Christopher

PBPA from Mycobacterium tuberculosis is a class B-like penicillin-binding protein (PBP) that is not essential for cell growth in M. tuberculosis, but is important for proper cell division in Mycobacterium smegmatis. We have determined the crystal structure of PBPA at 2.05 {angstrom} resolution, the first published structure of a PBP from this important pathogen. Compared to other PBPs, PBPA has a relatively small N-terminal domain, and conservation of a cluster of charged residues within this domain suggests that PBPA is more related to class B PBPs than previously inferred from sequence analysis. The C-terminal domain is a typical transpeptidase foldmore » and contains the three conserved active-site motifs characterisitic of penicillin-interacting enzymes. While the arrangement of the SxxK and KTG motifs is similar to that observed in other PBPs, the SxN motif is markedly displaced away from the active site, such that its serine (Ser281) is not involved in hydrogen bonding with residues of the other two motifs. A disulfide bridge between Cys282 (the 'x' of the SxN motif) and Cys266, which resides on an adjacent loop, may be responsible for this unusual conformation. Another interesting feature of the structure is a relatively long connection between {beta}5 and {alpha}11, which restricts the space available in the active site of PBPA and suggests that conformational changes would be required to accommodate peptide substrate or {beta}-lactam antibiotics during acylation. Finally, the structure shows that one of the two threonines postulated to be targets for phosphorylation is inaccessible (Thr362), whereas the other (Thr437) is well placed on a surface loop near the active site.« less

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification

PubMed Central

Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida

2017-01-01

Abstract Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. PMID:28053125

Soil Viral Communities Vary Temporally and along a Land Use Transect as Revealed by Virus-Like Particle Counting and a Modified Community Fingerprinting Approach (fRAPD)

PubMed Central

Narr, Anja; Nawaz, Ali; Wick, Lukas Y.; Harms, Hauke; Chatzinotas, Antonis

2017-01-01

Environmental surveys on soil viruses are still rare and mostly anecdotal, i. e., they mostly report on viruses at one location or for only a few sampling dates. Detailed time-series analysis with multiple samples can reveal the spatio-temporal dynamics of viral communities and provide important input as to how viruses interact with their potential hosts and the environment. Such surveys, however, require fast, easy-to-apply and reliable methods. In the present study we surveyed monthly across 13 months the abundance of virus-like particles (VLP) and the structure of the viral communities in soils along a land use transect (i.e., forest, pasture, and cropland). We evaluated 32 procedures to extract VLP from soil using different buffers and mechanical methods. The most efficient extraction was achieved with 1× saline magnesium buffer in combination with 20 min vortexing. For community structure analysis we developed an optimized fingerprinting approach (fluorescent RAPD-PCR; fRAPD) by combining RAPD-PCR with fluorescently labeled primers in order to size the obtained fragments on a capillary sequencing machine. With the concomitantly collected data of soil specific factors and weather data, we were able to find correlations of viral abundance and community structure with environmental variables and sampling site. More specifically, we found that soil specific factors such as pH and total nitrogen content played a significant role in shaping both soil viral abundance and community structure. The fRAPD analysis revealed high temporal changes and clustered the viral communities according to sampling sites. In particular we observed that temperature and rainfall shaped soil viral communities in non-forest sites. In summary our findings suggest that sampling site was a key factor for shaping the abundance and community structure of soil viruses, and when site vegetation was reduced, temperature and rainfall were also important factors. PMID:29067022

Distance, dams and drift: What structures populations of an endangered, benthic stream fish?

USGS Publications Warehouse

Roberts, James H.; Angermeier, Paul; Hallerman, Eric M.

2013-01-01

Spatial population structure plays an important role in species persistence, evolution and conservation. Benthic stream fishes are diverse and frequently imperilled, yet the determinants and spatial scaling of their population structure are understudied. We investigated the range-wide population genetic structure of Roanoke logperch (Percina rex), an endangered, benthic stream fish of the eastern United States. Fish were sampled from 35 sites and analysed at 11 microsatellite DNA loci. Clustering models were used to sort individuals into genetically cohesive groups and thereby estimate the spatial scaling of population structure. We then used Bayesian generalized linear mixed models (BGLMMs) to test alternative hypotheses about the environmental factors most responsible for generating structure, as measured by the differentiation statistic FST. Clustering models delineated seven discrete populations, whose boundaries coincided with agents of fragmentation, including hydroelectric dams and tailwaters. In the absence of hydrological barriers, gene flow was extensive throughout catchments, whereas there was no evidence for contemporary dispersal between catchments across barriers. In the best-supported BGLMM, FST was positively related to the spatial distance and degree of hydrological alteration between sites and negatively related to genetic diversity within sites. Whereas the effect of tailwaters was equivocal, dams strongly influenced differentiation: the effect of a dam on FST was comparable to that of a between-site distance of over 1200 km of unimpounded river. Overall, the effect of distance-mediated dispersal was negligible compared to the combined effects of fragmentation and genetic drift. The contemporary population structure of P. rex comprises a few geographically extensive ‘islands’ that are fragmented by hydroelectric projects. This information clarifies the importance of a catchment-scale perspective on conserving the species and suggests that its recovery may require genetic and/or demographic reconnection of presently isolated populations.

Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain.

PubMed

Sriram, M; Osipiuk, J; Freeman, B; Morimoto, R; Joachimiak, A

1997-03-15

The 70 kDa heat shock proteins (Hsp70) are a family of molecular chaperones, which promote protein folding and participate in many cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whereas the C-terminal domain is required for polypeptide binding. Cooperation of both domains is needed for protein folding. The crystal structure of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch are functionally linked with ATP hydrolysis. A high-resolution structure of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C- and N-terminal domains. The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1. 84 A, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridging ADP and inorganic phosphate, and the second calcium is tightly coordinated on the protein surface by Glu231, Asp232 and the carbonyl of His227. Overall, the structure of hATPase is similar to bATPase. Differences between them are found in the loops, the sites of amino acid substitution and the calcium-binding sites. Human Hsp70 chaperone is phosphorylated in vitro in the presence of divalent ions, calcium being the most effective. The structural similarity of hATPase and bATPase and the sequence similarity within the Hsp70 chaperone family suggest a universal mechanism of ATP hydrolysis among all Hsp70 molecular chaperones. Two calcium ions have been found in the hATPase structure. One corresponds to the magnesium site in bATPase and appears to be important for ATP hydrolysis and in vitro phosphorylation. Local changes in protein structure as a result of calcium binding may facilitate phosphorylation. A small, but significant, movement of metal ions and sidechains could position catalytically important threonine residues for phosphorylation. The second calcium site represents a new calcium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in the active site could be transmitted to the peptide-binding domain.

Development Directions for Various Types of the Light Wood-Framed Structures

NASA Astrophysics Data System (ADS)

Malesza, J.; Baszeń, M.; Miedziałowski, Cz

2017-11-01

The paper presents current trends in the development of the wood-framed structures. Authors describe the evolution of the technology of implementation, the production process of precast elements of buildings as well as selected realization on the site of these kinds of structures. The attention has been paid to the effect of implementation phases on construction and erecting technology of the wood-framed structures. The paper draws attention to the importance and enhancement of structural analysis of structures in individual phases of building realization.

Relationships between Algae, Benthic Herbivorous Invertebrates and Fishes in Rocky Sublittoral Communities of a Temperate Sea (Mediterranean)

NASA Astrophysics Data System (ADS)

Ruitton, S.; Francour, P.; Boudouresque, C. F.

2000-02-01

In situ surveys were used to examine the contribution of benthic herbivorous invertebrates and fishes to the organization of Mediterranean rocky sublittoral communities. Shallow (1-3 m) and deep (6-8 m) sampling sites, in natural areas and on man-made structures, were characterized by a structural complexity index (cavity index and mean size of cavity aperture), algal cover (encrusting, turfy, shrubby and arborescent algae) and the density of benthic herbivorous invertebrates and fish. A relationship between structural complexity and biota was only evident for some fish species ( Diplodus spp. and Sarpa salpa) at deep sites, where they not only feed but also shelter. Three benthic herbivorous invertebrates, the sea urchins Paracentrotus lividus and Arbacia lixula, and the limpet Patella caerulea , are associated with communities dominated by encrusting algae. Variations in their abundance and role in structuring algal communities follow a depth gradient: P. caerulea and A. lixula are mainly present at shallow sites and P. lividus at deep sites. These benthic herbivorous invertebrates may account for the structure of shallow algal communities. In contrast, at deep sites, fishes (the omnivorous Diplodus spp. and the herbivorous S. salpa) have a potential importance in controlling sublittoral algae, in addition to invertebrates. It is suggested that the ecological impact of herbivorous and omnivorous fishes in temperate seas could be greater than is generally thought. Experiments should be designed to validate this postulate.

Structural study of the Fox-1 RRM protein hydration reveals a role for key water molecules in RRM-RNA recognition

PubMed Central

Blatter, Markus; Cléry, Antoine; Damberger, Fred F.

2017-01-01

Abstract The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes. PMID:28505313

Structural evidence for a copper-bound carbonate intermediate in the peroxidase and dismutase activities of superoxide dismutase.

PubMed

Strange, Richard W; Hough, Michael A; Antonyuk, Svetlana V; Hasnain, S Samar

2012-01-01

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O(2) and H(2)O(2). SOD also reacts with H(2)O(2), leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO(2)) and H(2)O(2), this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

PubMed Central

Strange, Richard W.; Hough, Michael A.; Antonyuk, Svetlana V.; Hasnain, S. Samar

2012-01-01

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O2 and H2O2. SOD also reacts with H2O2, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO2) and H2O2, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper. PMID:22984565

Influence of corn, switchgrass, and prairie cropping systems on soil microbial communities in the upper Midwest of the United States

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jesus, Ederson da C.; Liang, Chao; Quensen, John F.

Because soil microbes drive many of the processes underpinning ecosystem services provided by soils, understanding how cropping systems affect soil microbial communities is important for productive and sustainable management. We characterized and compared soil microbial communities under restored prairie and three potential cellulosic biomass crops (corn, switchgrass, and mixed prairie grasses) in two spatial experimental designs – side-by-side plots where plant communities were in their second year since establishment (i.e., intensive sites) and regionally distributed fields where plant communities had been in place for at least 10 years (i.e., extensive sites). We assessed microbial community structure and composition using lipidmore » analysis, pyrosequencing of rRNA genes (targeting fungi, bacteria, archaea, and lower eukaryotes), and targeted metagenomics of nifH genes. For the more recently established intensive sites, soil type was more important than plant community in determining microbial community structure, while plant community was the more important driver of soil microbial communities for the older extensive sites where microbial communities under corn were clearly differentiated from those under switchgrass and restored prairie. Here, bacterial and fungal biomasses, especially biomass of arbuscular mycorrhizal fungi, were higher under perennial grasses and restored prairie, suggesting a more active carbon pool and greater microbial processing potential, which should be beneficial for plant acquisition and ecosystem retention of carbon, water, and nutrients.« less

Influence of corn, switchgrass, and prairie cropping systems on soil microbial communities in the upper Midwest of the United States

DOE PAGES

Jesus, Ederson da C.; Liang, Chao; Quensen, John F.; ...

2015-06-28

Because soil microbes drive many of the processes underpinning ecosystem services provided by soils, understanding how cropping systems affect soil microbial communities is important for productive and sustainable management. We characterized and compared soil microbial communities under restored prairie and three potential cellulosic biomass crops (corn, switchgrass, and mixed prairie grasses) in two spatial experimental designs – side-by-side plots where plant communities were in their second year since establishment (i.e., intensive sites) and regionally distributed fields where plant communities had been in place for at least 10 years (i.e., extensive sites). We assessed microbial community structure and composition using lipidmore » analysis, pyrosequencing of rRNA genes (targeting fungi, bacteria, archaea, and lower eukaryotes), and targeted metagenomics of nifH genes. For the more recently established intensive sites, soil type was more important than plant community in determining microbial community structure, while plant community was the more important driver of soil microbial communities for the older extensive sites where microbial communities under corn were clearly differentiated from those under switchgrass and restored prairie. Here, bacterial and fungal biomasses, especially biomass of arbuscular mycorrhizal fungi, were higher under perennial grasses and restored prairie, suggesting a more active carbon pool and greater microbial processing potential, which should be beneficial for plant acquisition and ecosystem retention of carbon, water, and nutrients.« less

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families.

PubMed

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Svedas, Vytas

2014-07-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure-function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl.EDTA and localization of the major dye binding sites.

PubMed Central

Tanner, N K; Cech, T R

1985-01-01

The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymena thermophila is converted to a covalently closed circular RNA in the absence of proteins in vitro. This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl.EDTA (MPE; R.P. Hertzberg and P.B. Dervan (1982) J. Am. Chem. Soc. 104, 313-315). The MPE binding sites have been localized by mapping the sites of MPE.Fe(II) cleavage of the IVS RNA. There are three major binding sites within the 414 nucleotide IVS RNA. Two of these sites coincide with the A.B and 9L.2 pairings. These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing. We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization. The reactions of MPE.Fe(II) with an RNA of known structure, tRNAPhe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration. Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule. Images PMID:2415924

The effect of glycosylation on the transferrin structure: A molecular dynamic simulation analysis.

PubMed

Ghanbari, Z; Housaindokht, M R; Bozorgmehr, M R; Izadyar, M

2016-09-07

Transferrins have been defined by the highly cooperative binding of iron and a carbonate anion to form a Fe-CO3-Tf ternary complex. As such, the layout of the binding site residues affects transferrin function significantly; In contrast to N-lobe, C-lobe binding site of the transferrin structure has been less characterized and little research which surveyed the interaction of carbonate with transferrin in the C-lobe binding site has been found. In the present work, molecular dynamic simulation was employed to gain access into the molecular level understanding of carbonate binding site and their interactions in each lobe. Residues responsible for carbonate binding of transferrin structure were pointed out. In addition, native human transferrin is a glycoprotein that two N-linked complex glycan chains located in the C-lobe. Usually, in the molecular dynamic simulation for simplifying, glycan is removed from the protein structure. Here, we explore the effect of glycosylation on the transferrin structure. Glycosylation appears to have an effect on the layout of the binding site residue and transferrin structure. On the other hand, sometimes the entire transferrin formed by separated lobes that it allows the results to be interpreted in a straightforward manner rather than more parameters required for full length protein. But, it should be noted that there are differences between the separated lobe and full length transferrin, hence, a comparative analysis by the molecular dynamic simulation was performed to investigate such structural variations. Results revealed that separation in C-lobe caused a significant structural variation in comparison to N-lobe. Consequently, the separated lobes and the full length one are different, showing the importance of the interlobe communication and the impact of the lobes on each other in the transferrin structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

PubMed Central

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-01-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

POOL server: machine learning application for functional site prediction in proteins.

PubMed

Somarowthu, Srinivas; Ondrechen, Mary Jo

2012-08-01

We present an automated web server for partial order optimum likelihood (POOL), a machine learning application that combines computed electrostatic and geometric information for high-performance prediction of catalytic residues from 3D structures. Input features consist of THEMATICS electrostatics data and pocket information from ConCavity. THEMATICS measures deviation from typical, sigmoidal titration behavior to identify functionally important residues and ConCavity identifies binding pockets by analyzing the surface geometry of protein structures. Both THEMATICS and ConCavity (structure only) do not require the query protein to have any sequence or structure similarity to other proteins. Hence, POOL is applicable to proteins with novel folds and engineered proteins. As an additional option for cases where sequence homologues are available, users can include evolutionary information from INTREPID for enhanced accuracy in site prediction. The web site is free and open to all users with no login requirements at http://www.pool.neu.edu. m.ondrechen@neu.edu Supplementary data are available at Bioinformatics online.

A Bridge between Worlds: Understanding Network Structure to Understand Change Strategy

ERIC Educational Resources Information Center

Daly, Alan J.; Finnigan, Kara S.

2010-01-01

A number of scholars are exploring district and site relations in organizational change efforts in the larger policy context of No Child Left Behind. These studies suggest the importance of the central office as a support to the work of reform and offer strategies for building relations between district offices and sites in order to implement and…

Coral identity underpins architectural complexity on Caribbean reefs.

PubMed

Alvarez-Filip, Lorenzo; Dulvy, Nicholas K; Côte, Isabelle M; Watkinson, Andrew R; Gill, Jennifer A

2011-09-01

The architectural complexity of ecosystems can greatly influence their capacity to support biodiversity and deliver ecosystem services. Understanding the components underlying this complexity can aid the development of effective strategies for ecosystem conservation. Caribbean coral reefs support and protect millions of livelihoods, but recent anthropogenic change is shifting communities toward reefs dominated by stress-resistant coral species, which are often less architecturally complex. With the regionwide decline in reef fish abundance, it is becoming increasingly important to understand changes in coral reef community structure and function. We quantify the influence of coral composition, diversity, and morpho-functional traits on the architectural complexity of reefs across 91 sites at Cozumel, Mexico. Although reef architectural complexity increases with coral cover and species richness, it is highest on sites that are low in taxonomic evenness and dominated by morpho-functionally important, reef-building coral genera, particularly Montastraea. Sites with similar coral community composition also tend to occur on reefs with very similar architectural complexity, suggesting that reef structure tends to be determined by the same key species across sites. Our findings provide support for prioritizing and protecting particular reef types, especially those dominated by key reef-building corals, in order to enhance reef complexity.

Seismically induced liquefaction structures in La Magdalena archaeological site, the 4th century AD Roman Complutum (Madrid, Spain)

NASA Astrophysics Data System (ADS)

Rodríguez-Pascua, M. A.; Silva, P. G.; Perucha, M. A.; Giner-Robles, J. L.; Heras, C.; Bastida, A. B.; Carrasco, P.; Roquero, E.; Lario, J.; Bardaji, T.; Pérez-López, R.; Elez, J.

2016-10-01

The ancient Roman city of Complutum (Alcalá de Henares, Madrid), founded in the 1st century AD, was one of the most important cities of Hispania. The old Roman city was destroyed, abruptly abandoned, relocated close by and rebuilt during the late 4th century AD. Destruction of the city and its relocation has not yet been explained by archaeologists. In this paper, with our multidisciplinary approach, we identify and characterize earthquake archaeological effects (EAEs) affecting the archaeological site, the La Magdalena, an agricultural holding 4 km from the core of Complutum. The most important EAEs in the site are liquefactions (sand dikes and explosive sand-gravel craters) affecting Roman structures, such as water tanks (cisterns), houses and graves. Ground liquefaction generated significant ground cracks, explosive craters and folds in foundations of buildings. Several other Roman sites throughout the valley were also abandoned abruptly during the 4th century AD, in some cases with EAEs of similar origin. This suggests the occurrence of a 5.0-6.6 Mw seismic event in the zone, in accordance with the minimum empirical limit of seismically-induced liquefaction and the maximum surface rupture length of the Henares fault.

Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii.

PubMed

Pampa, K J; Madan Kumar, S; Hema, M K; Kumara, Karthik; Naveen, S; Kunishima, Naoki; Lokanath, N K

2017-12-01

Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-L-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subunits, presumably near to the active site(s) of the enzyme. The observed dimeric state might be important for the catalytic function of the enzyme.

Web-ware bioinformatical analysis and structure modelling of N-terminus of human multisynthetase complex auxiliary component protein p43.

PubMed

Deineko, Viktor

2006-01-01

Human multisynthetase complex auxiliary component, protein p43 is an endothelial monocyte-activating polypeptide II precursor. In this study, comprehensive sequence analysis of N-terminus has been performed to identify structural domains, motifs, sites of post-translation modification and other functionally important parameters. The spatial structure model of full-chain protein p43 is obtained.

REGIONAL, WATERSHED, AND SITE-SPECIFIC ENVIRONMENTAL INFLUENCES ON FISH ASSEMBLAGE STRUCTURE AND FUNCTION IN WESTERN LAKE SUPERIOR TRIBUTARIES

EPA Science Inventory

The relative importance of regional, watershed, and in-stream environmental factors on stream fish assemblage structure and function was investigated as part of a comparative watershed project in the western Lake Superior basin. We selected 48 second and third order watersheds fr...

Simulating Food Web Dynamics along a Gradient: Quantifying Human Influence

PubMed Central

Jordán, Ferenc; Gjata, Nerta; Mei, Shu; Yule, Catherine M.

2012-01-01

Realistically parameterized and dynamically simulated food-webs are useful tool to explore the importance of the functional diversity of ecosystems, and in particular relations between the dynamics of species and the whole community. We present a stochastic dynamical food web simulation for the Kelian River (Borneo). The food web was constructed for six different locations, arrayed along a gradient of increasing human perturbation (mostly resulting from gold mining activities) along the river. Along the river, the relative importance of grazers, filterers and shredders decreases with increasing disturbance downstream, while predators become more dominant in governing eco-dynamics. Human activity led to increased turbidity and sedimentation which adversely impacts primary productivity. Since the main difference between the study sites was not the composition of the food webs (structure is quite similar) but the strengths of interactions and the abundance of the trophic groups, a dynamical simulation approach seemed to be useful to better explain human influence. In the pristine river (study site 1), when comparing a structural version of our model with the dynamical model we found that structurally central groups such as omnivores and carnivores were not the most important ones dynamically. Instead, primary consumers such as invertebrate grazers and shredders generated a greater dynamical response. Based on the dynamically most important groups, bottom-up control is replaced by the predominant top-down control regime as distance downstream and human disturbance increased. An important finding, potentially explaining the poor structure to dynamics relationship, is that indirect effects are at least as important as direct ones during the simulations. We suggest that our approach and this simulation framework could serve systems-based conservation efforts. Quantitative indicators on the relative importance of trophic groups and the mechanistic modeling of eco-dynamics could greatly contribute to understanding various aspects of functional diversity. PMID:22768346

Structure of the mouse galectin-4 N-terminal carbohydrate-recognition domain reveals the mechanism of oligosaccharide recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krejciríková, Veronika; Pachl, Petr; Fábry, Milan

2011-11-18

Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 {angstrom} resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K{sub d} value in the micromolar range (K{submore » d1} = 600 {+-} 70 {mu}M) and a low-affinity site with K{sub d2} = 28 {+-} 10 mM.« less

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

PubMed

Xu, Xiang; Zhao, Jingyue; Xu, Zhen; Peng, Baozhen; Huang, Qiuhua; Arnold, Eddy; Ding, Jianping

2004-08-06

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Structural Insights into E. coli Porphobilinogen Deaminase during Synthesis and Exit of 1-Hydroxymethylbilane

PubMed Central

Bulusu, Gopalakrishnan

2014-01-01

Porphobilinogen deaminase (PBGD) catalyzes the formation of 1-hydroxymethylbilane (HMB), a crucial intermediate in tetrapyrrole biosynthesis, through a step-wise polymerization of four molecules of porphobilinogen (PBG), using a unique dipyrromethane (DPM) cofactor. Structural and biochemical studies have suggested residues with catalytic importance, but their specific role in the mechanism and the dynamic behavior of the protein with respect to the growing pyrrole chain remains unknown. Molecular dynamics simulations of the protein through the different stages of pyrrole chain elongation suggested that the compactness of the overall protein decreases progressively with addition of each pyrrole ring. Essential dynamics showed that domains move apart while the cofactor turn region moves towards the second domain, thus creating space for the pyrrole rings added at each stage. Residues of the flexible active site loop play a significant role in its modulation. Steered molecular dynamics was performed to predict the exit mechanism of HMB from PBGD at the end of the catalytic cycle. Based on the force profile and minimal structural changes the proposed path for the exit of HMB is through the space between the domains flanking the active site loop. Residues reported as catalytically important, also play an important role in the exit of HMB. Further, upon removal of HMB, the structure of PBGD gradually relaxes to resemble its initial stage structure, indicating its readiness to resume a new catalytic cycle. PMID:24603363

Molecular Evolution of Ultraspiracle Protein (USP/RXR) in Insects

PubMed Central

Hult, Ekaterina F.; Tobe, Stephen S.; Chang, Belinda S. W.

2011-01-01

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (d N/d S), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that d S is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand. PMID:21901121

Long-lived groupers require structurally stable reefs in the face of repeated climate change disturbances

NASA Astrophysics Data System (ADS)

Karkarey, R.; Kelkar, N.; Lobo, A. Savio; Alcoverro, T.; Arthur, R.

2014-06-01

Benthic recovery from climate-related disturbances does not always warrant a commensurate functional recovery for reef-associated fish communities. Here, we examine the distribution of benthic groupers (family Serranidae) in coral reef communities from the Lakshadweep archipelago (Arabian Sea) in response to structural complexity and long-term habitat stability. These coral reefs that have been subject to two major El Niño Southern Oscillation-related coral bleaching events in the last decades (1998 and 2010). First, we employ a long-term (12-yr) benthic-monitoring dataset to track habitat structural stability at twelve reef sites in the archipelago. Structural stability of reefs was strongly driven by exposure to monsoon storms and depth, which made deeper and more sheltered reefs on the eastern aspect more stable than the more exposed (western) and shallower reefs. We surveyed groupers (species richness, abundance, biomass) in 60 sites across the entire archipelago, representing both exposures and depths. Sites were selected along a gradient of structural complexity from very low to high. Grouper biomass appeared to vary with habitat stability with significant differences between depth and exposure; sheltered deep reefs had a higher grouper biomass than either sheltered shallow or exposed (deep and shallow) reefs. Species richness and abundance showed similar (though not significant) trends. More interestingly, average grouper biomass increased exponentially with structural complexity, but only at the sheltered deep (high stability) sites, despite the availability of recovered structure at exposed deep and shallow sites (lower-stability sites). This trend was especially pronounced for long-lived groupers (life span >10 yrs). These results suggest that long-lived groupers may prefer temporally stable reefs, independent of the local availability of habitat structure. In reefs subject to repeated disturbances, the presence of structurally stable reefs may be critical as refuges for functionally important, long-lived species like groupers.

Modelling forest canopy height by integrating airborne LiDAR samples with satellite Radar and multispectral imagery

NASA Astrophysics Data System (ADS)

García, Mariano; Saatchi, Sassan; Ustin, Susan; Balzter, Heiko

2018-04-01

Spatially-explicit information on forest structure is paramount to estimating aboveground carbon stocks for designing sustainable forest management strategies and mitigating greenhouse gas emissions from deforestation and forest degradation. LiDAR measurements provide samples of forest structure that must be integrated with satellite imagery to predict and to map landscape scale variations of forest structure. Here we evaluate the capability of existing satellite synthetic aperture radar (SAR) with multispectral data to estimate forest canopy height over five study sites across two biomes in North America, namely temperate broadleaf and mixed forests and temperate coniferous forests. Pixel size affected the modelling results, with an improvement in model performance as pixel resolution coarsened from 25 m to 100 m. Likewise, the sample size was an important factor in the uncertainty of height prediction using the Support Vector Machine modelling approach. Larger sample size yielded better results but the improvement stabilised when the sample size reached approximately 10% of the study area. We also evaluated the impact of surface moisture (soil and vegetation moisture) on the modelling approach. Whereas the impact of surface moisture had a moderate effect on the proportion of the variance explained by the model (up to 14%), its impact was more evident in the bias of the models with bias reaching values up to 4 m. Averaging the incidence angle corrected radar backscatter coefficient (γ°) reduced the impact of surface moisture on the models and improved their performance at all study sites, with R2 ranging between 0.61 and 0.82, RMSE between 2.02 and 5.64 and bias between 0.02 and -0.06, respectively, at 100 m spatial resolution. An evaluation of the relative importance of the variables in the model performance showed that for the study sites located within the temperate broadleaf and mixed forests biome ALOS-PALSAR HV polarised backscatter was the most important variable, with Landsat Tasselled Cap Transformation components barely contributing to the models for two of the study sites whereas it had a significant contribution at the third one. Over the temperate conifer forests, Landsat Tasselled Cap variables contributed more than the ALOS-PALSAR HV band to predict the landscape height variability. In all cases, incorporation of multispectral data improved the retrieval of forest canopy height and reduced the estimation uncertainty for tall forests. Finally, we concluded that models trained at one study site had higher uncertainty when applied to other sites, but a model developed from multiple sites performed equally to site-specific models to predict forest canopy height. This result suggest that a biome level model developed from several study sites can be used as a reliable estimator of biome-level forest structure from existing satellite imagery.

A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by its dodecameric structure

PubMed Central

Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A.

2015-01-01

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key towards understanding the regulation of polyamine levels in bacteria during pathogenesis. PMID:25623305

Crystal Structure of Cockroach Allergen Bla g 2, an Unusual Zinc Binding Aspartic Protease with a Novel Mode of Self-inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gustchina, Alla; Li, Mi; Wunschmann, Sabina

2010-07-19

The crystal structure of Bla g 2 was solved in order to investigate the structural basis for the allergenic properties of this unusual protein. This is the first structure of an aspartic protease in which conserved glycine residues, in two canonical DTG triads, are substituted by different amino acid residues. Another unprecedented feature revealed by the structure is the single phenylalanine residue insertion on the tip of the flap, with the side-chain occupying the S1 binding pocket. This and other important amino acid substitutions in the active site region of Bla g 2 modify the interactions in the vicinity ofmore » the catalytic aspartate residues, increasing the distance between them to {approx}4 {angstrom} and establishing unique direct contacts between the flap and the catalytic residues. We attribute the absence of substantial catalytic activity in Bla g 2 to these unusual features of the active site. Five disulfide bridges and a Zn-binding site confer stability to the protein, which may contribute to sensitization at lower levels of exposure than other allergens.« less

The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode†

PubMed Central

Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

2013-01-01

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation. PMID:23735230

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Effects of floods on fish assemblages in an intermittent prairie stream

USGS Publications Warehouse

Franssen, N.R.; Gido, K.B.; Guy, C.S.; Tripe, J.A.; Shrank, S.J.; Strakosh, T.R.; Bertrand, K.N.; Franssen, C.M.; Pitts, K.L.; Paukert, C.P.

2006-01-01

1. Floods are major disturbances to stream ecosystems that can kill or displace organisms and modify habitats. Many studies have reported changes in fish assemblages after a single flood, but few studies have evaluated the importance of timing and intensity of floods on long-term fish assemblage dynamics. 2. We used a 10-year dataset to evaluate the effects of floods on fishes in Kings Creek, an intermittent prairie stream in north-eastern, Kansas, U.S.A. Samples were collected seasonally at two perennial headwater sites (1995-2005) and one perennial downstream flowing site (1997-2005) allowing us to evaluate the effects of floods at different locations within a watershed. In addition, four surveys during 2003 and 2004 sampled 3-5 km of stream between the long-term study sites to evaluate the use of intermittent reaches of this stream. 3. Because of higher discharge and bed scouring at the downstream site, we predicted that the fish assemblage would have lowered species richness and abundance following floods. In contrast, we expected increased species richness and abundance at headwater sites because floods increase stream connectivity and create the potential for colonisation from downstream reaches. 4. Akaike Information Criteria (AIC) was used to select among candidate regression models that predicted species richness and abundance based on Julian date, time since floods, season and physical habitat at each site. At the downstream site, AIC weightings suggested Julian date was the best predictor of fish assemblage structure, but no model explained >16% of the variation in species richness or community structure. Variation explained by Julian date was primarily attributed to a long-term pattern of declining abundance of common species. At the headwater sites, there was not a single candidate model selected to predict total species abundance and assemblage structure. AIC weightings suggested variation in assemblage structure was associated with either Julian date or local habitat characteristics. 5. Fishes rapidly colonised isolated or dry habitats following floods. This was evidenced by the occurrence of fishes in intermittent reaches and the positive association between maximum daily discharge and colonisation events at both headwater sites. 6. Our study suggests floods allow dispersal into intermittent habitats with little or no downstream displacement of fishes. Movement of fishes among habitats during flooding highlights the importance of maintaining connectivity of stream networks of low to medium order prairie streams. ?? 2006 The Authors.

Structural explanation for the role of Mn2+ in the activity of phi6 RNA-dependent RNA polymerase.

PubMed

Poranen, Minna M; Salgado, Paula S; Koivunen, Minni R L; Wright, Sam; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2008-11-01

The biological role of manganese (Mn(2+)) has been a long-standing puzzle, since at low concentrations it activates several polymerases whilst at higher concentrations it inhibits. Viral RNA polymerases possess a common architecture, reminiscent of a closed right hand. The RNA-dependent RNA polymerase (RdRp) of bacteriophage 6 is one of the best understood examples of this important class of polymerases. We have probed the role of Mn(2+) by biochemical, biophysical and structural analyses of the wild-type enzyme and of a mutant form with an altered Mn(2+)-binding site (E491 to Q). The E491Q mutant has much reduced affinity for Mn(2+), reduced RNA binding and a compromised elongation rate. Loss of Mn(2+) binding structurally stabilizes the enzyme. These data and a re-examination of the structures of other viral RNA polymerases clarify the role of manganese in the activation of polymerization: Mn(2+) coordination of a catalytic aspartate is necessary to allow the active site to properly engage with the triphosphates of the incoming NTPs. The structural flexibility caused by Mn(2+) is also important for the enzyme dynamics, explaining the requirement for manganese throughout RNA polymerization.

Full-scale monitoring of wind and suspension bridge response

NASA Astrophysics Data System (ADS)

Snæbjörnsson, J. T.; Jakobsen, J. B.; Cheynet, E.; Wang, J.

2017-12-01

Monitoring of real structures is important for many reasons. For structures susceptible to environmental actions, full-scale observations can provide valuable information about the environmental conditions at the site, as well as the characteristics of the excitation acting on the structure. The recorded data, if properly analyzed, can be used to validate and/or update experiments and models used in the design of new structures, such as the load description and modelling of the structural response. Various aspects of full-scale monitoring are discussed in the paper and the full-scale wind engineering laboratory at the Lysefjord suspension bridge introduced. The natural excitation of the bridge comes from wind and traffic. The surrounding terrain is complex and its effect on the wind flow can only be fully studied on site, in full-scale. The monitoring program and associated data analysis are described. These include various studies of the relevant turbulence characteristics, identification of dynamic properties and estimation of wind- and traffic-induced response parameters. The overall monitoring activity also included a novel application of the remote optical sensing in bridge engineering, which is found to have an important potential to complement traditional “single-point” wind observations by sonic anemometers.

Genetic mapping uncovers cis-regulatory landscape of RNA editing.

PubMed

Ramaswami, Gokul; Deng, Patricia; Zhang, Rui; Anna Carbone, Mary; Mackay, Trudy F C; Li, Jin Billy

2015-09-16

Adenosine-to-inosine (A-to-I) RNA editing, catalysed by ADAR enzymes conserved in metazoans, plays an important role in neurological functions. Although the fine-tuning mechanism provided by A-to-I RNA editing is important, the underlying rules governing ADAR substrate recognition are not well understood. We apply a quantitative trait loci (QTL) mapping approach to identify genetic variants associated with variability in RNA editing. With very accurate measurement of RNA editing levels at 789 sites in 131 Drosophila melanogaster strains, here we identify 545 editing QTLs (edQTLs) associated with differences in RNA editing. We demonstrate that many edQTLs can act through changes in the local secondary structure for edited dsRNAs. Furthermore, we find that edQTLs located outside of the edited dsRNA duplex are enriched in secondary structure, suggesting that distal dsRNA structure beyond the editing site duplex affects RNA editing efficiency. Our work will facilitate the understanding of the cis-regulatory code of RNA editing.

Properties of Skin in Chinese Infants: Developmental Changes in Ceramides and in Protein Secondary Structure of the Stratum Corneum

PubMed Central

Yuan, Chao; Zou, Ying; Xueqiu, Yao; Miyauchi, Yuki; Fujimura, Tsutomu; Kitahara, Takashi; Wang, Xuemin

2017-01-01

The properties of infant skin regarding its structure and stratum corneum (SC) properties during development compared to adult skin have been reported only for a few races and body sites. The aim of this study was to understand the developmental changes of skin properties in Chinese infants, focusing on SC ceramides and protein secondary structure, which are important for skin barrier function. Three body sites with distinct characteristics (cheeks, inner upper arms, and buttocks) were assessed. Sixty pairs of Chinese infants and their mothers were measured for SC hydration, transepidermal water loss, ceramide levels, sebum with an ester bond, and protein secondary structure of superficial SC. Skin hydration decreased with age at all body sites. TEWL was similar between the 2–12- and 13–24-month-old groups but was higher than the adult group at the buttocks and inner upper arms and was equal to the adult group at the cheeks. These differences coincided with differences in protein secondary structure. Ceramide and sebum levels were lower in the infant groups. We conclude that both the SC functions and the components of infant skin are still developing and are not fully adapted as in adult skin at each body site examined. PMID:29098152

Structural studies of the rhombohedral and orthorhombic monouranates: CaUO{sub 4}, α-SrUO{sub 4}, β-SrUO{sub 4} and BaUO{sub 4}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Gabriel; Kennedy, Brendan J., E-mail: kennedyb@chem.usyd.edu.au; Johannessen, Bernt

The structures of some AUO{sub 4} (A=Ca, Sr, or Ba) oxides have been determined using a combination of neutron and synchrotron X-ray diffraction, supported by X-ray absorption spectroscopic measurements at the U L{sub 3}-edge. The smaller Ca cation favours a rhombohedral AUO{sub 4} structure with 8-coordinate UO{sub 8} moieties whilst an orthorhombic structure based on UO{sub 6} groups is found for BaUO{sub 4}. Both the rhombohedral and orthorhombic structures can be stabilised for SrUO{sub 4}. The structural studies suggest that the bonding requirements of the A site cation play a significant role in determining which structure is favoured. In themore » rhombohedral structure, Bond Valence Sums demonstrate the A site is invariably overbonded, which, in the case of rhombohedral α-SrUO{sub 4}, is compensated for by the formation of vacancies in the oxygen sub-lattice. The uranium cation, with its flexible oxidation state, is able to accommodate this by inducing vacancies along its equatorial coordination site as demonstrated by neutron powder diffraction. - Graphical abstract: Diffraction studies of AUO{sub 4} (A = Ca, Sr, or Ba) oxides reveal the importance of the bonding requirements of the A site cation in determining whether the structure is rhombohedral or orthorhombic. - Highlights: • Structures of AUO{sub 4} ( A = Ca Sr, Ba) refined against X-ray and Neutron diffraction. • The alkali cations size has a dramatic effect on the crystal structure. • Smaller cations favouring a rhombohedral structure. • Oxygen vacancies to stabilise the rhombohedral structure in SrUO{sub 4}.« less

Relative importance of top-down and bottom-up forces in food webs of Sarracenia pitcher communities at a northern and a southern site.

PubMed

Hoekman, David

2011-04-01

The relative importance of resources (bottom-up forces) and natural enemies (top-down forces) for regulating food web dynamics has been debated, and both forces have been found to be critical for determining food web structure. How the relative importance of top-down and bottom-up forces varies between sites with different abiotic conditions is not well understood. Using the pitcher plant inquiline community as a model system, I examine how the relative importance of top-down and bottom-up effects differs between two disparate sites. Resources (ant carcasses) and top predators (mosquito larvae) were manipulated in two identical 4 × 4 factorial press experiments, conducted at two geographically distant sites (Michigan and Florida) within the range of the purple pitcher plant, Sarracenia purpurea, and the aquatic community that resides in its leaves. Overall, top predators reduced the density of prey populations while additional resources bolstered them, and the relative importance of top-down and bottom-up forces varied between sites and for different trophic levels. Specifically, top-down effects on protozoa were stronger in Florida than in Michigan, while the opposite pattern was found for rotifers. These findings experimentally demonstrate that the strength of predator-prey interactions, even those involving the same species, vary across space. While only two sites are compared in this study, I hypothesize that site differences in temperature, which influences metabolic rate, may be responsible for variation in consumer-resource interactions. These findings warrant further investigation into the specific factors that modify the relative importance of top-down and bottom-up effects.

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

NASA Astrophysics Data System (ADS)

Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

2017-03-01

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

How Does Salinity Shape Bacterial and Fungal Microbiomes of Alnus glutinosa Roots?

PubMed Central

Thiem, Dominika; Gołębiewski, Marcin; Hulisz, Piotr; Piernik, Agnieszka; Hrynkiewicz, Katarzyna

2018-01-01

Black alder (Alnus glutinosa Gaertn.) belongs to dual mycorrhizal trees, forming ectomycorrhizal (EM) and arbuscular (AM) root structures, as well as represents actinorrhizal plants that associate with nitrogen-fixing actinomycete Frankia sp. We hypothesized that the unique ternary structure of symbionts can influence community structure of other plant-associated microorganisms (bacterial and fungal endophytes), particularly under seasonally changing salinity in A. glutinosa roots. In our study we analyzed black alder root bacterial and fungal microbiome present at two forest test sites (saline and non-saline) in two different seasons (spring and fall). The dominant type of root microsymbionts of alder were ectomycorrhizal fungi, whose distribution depended on site (salinity): Tomentella, Lactarius, and Phialocephala were more abundant at the saline site. Mortierella and Naucoria (representatives of saprotrophs or endophytes) displayed the opposite tendency. Arbuscular mycorrhizal fungi belonged to Glomeromycota (orders Paraglomales and Glomales), however, they represented less than 1% of all identified fungi. Bacterial community structure depended on test site but not on season. Sequences affiliated with Rhodanobacter, Granulicella, and Sphingomonas dominated at the saline site, while Bradyrhizobium and Rhizobium were more abundant at the non-saline site. Moreover, genus Frankia was observed only at the saline site. In conclusion, bacterial and fungal community structure of alder root microsymbionts and endophytes depends on five soil chemical parameters: salinity, phosphorus, pH, saturation percentage (SP) as well as total organic carbon (TOC), and seasonality does not appear to be an important factor shaping microbial communities. Ectomycorrhizal fungi are the most abundant symbionts of mature alders growing in saline soils. However, specific distribution of nitrogen-fixing Frankia (forming root nodules) and association of arbuscular fungi at early stages of plant development should be taken into account in further studies. PMID:29720967

How Does Salinity Shape Bacterial and Fungal Microbiomes of Alnus glutinosa Roots?

PubMed

Thiem, Dominika; Gołębiewski, Marcin; Hulisz, Piotr; Piernik, Agnieszka; Hrynkiewicz, Katarzyna

2018-01-01

Black alder ( Alnus glutinosa Gaertn.) belongs to dual mycorrhizal trees, forming ectomycorrhizal (EM) and arbuscular (AM) root structures, as well as represents actinorrhizal plants that associate with nitrogen-fixing actinomycete Frankia sp. We hypothesized that the unique ternary structure of symbionts can influence community structure of other plant-associated microorganisms (bacterial and fungal endophytes), particularly under seasonally changing salinity in A. glutinosa roots. In our study we analyzed black alder root bacterial and fungal microbiome present at two forest test sites (saline and non-saline) in two different seasons (spring and fall). The dominant type of root microsymbionts of alder were ectomycorrhizal fungi, whose distribution depended on site (salinity): Tomentella , Lactarius , and Phialocephala were more abundant at the saline site. Mortierella and Naucoria (representatives of saprotrophs or endophytes) displayed the opposite tendency. Arbuscular mycorrhizal fungi belonged to Glomeromycota (orders Paraglomales and Glomales), however, they represented less than 1% of all identified fungi. Bacterial community structure depended on test site but not on season. Sequences affiliated with Rhodanobacter , Granulicella , and Sphingomonas dominated at the saline site, while Bradyrhizobium and Rhizobium were more abundant at the non-saline site. Moreover, genus Frankia was observed only at the saline site. In conclusion, bacterial and fungal community structure of alder root microsymbionts and endophytes depends on five soil chemical parameters: salinity, phosphorus, pH, saturation percentage (SP) as well as total organic carbon (TOC), and seasonality does not appear to be an important factor shaping microbial communities. Ectomycorrhizal fungi are the most abundant symbionts of mature alders growing in saline soils. However, specific distribution of nitrogen-fixing Frankia (forming root nodules) and association of arbuscular fungi at early stages of plant development should be taken into account in further studies.

Conserved nucleation sites reinforce the significance of Phi value analysis in protein-folding studies.

PubMed

Gianni, Stefano; Jemth, Per

2014-07-01

The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.

Accessibility and content of individualized adult reconstructive hip and knee/musculoskeletal oncology fellowship web sites.

PubMed

Young, Bradley L; Cantrell, Colin K; Patt, Joshua C; Ponce, Brent A

2018-06-01

Accessible, adequate online information is important to fellowship applicants. Program web sites can affect which programs applicants apply to, subsequently altering interview costs incurred by both parties and ultimately impacting rank lists. Web site analyses have been performed for all orthopaedic subspecialties other than those involved in the combined adult reconstruction and musculoskeletal (MSK) oncology fellowship match. A complete list of active programs was obtained from the official adult reconstruction and MSK oncology society web sites. Web site accessibility was assessed using a structured Google search. Accessible web sites were evaluated based on 21 previously reported content criteria. Seventy-four adult reconstruction programs and 11 MSK oncology programs were listed on the official society web sites. Web sites were identified and accessible for 58 (78%) adult reconstruction and 9 (82%) MSK oncology fellowship programs. No web site contained all content criteria and more than half of both adult reconstruction and MSK oncology web sites failed to include 12 of the 21 criteria. Several programs participating in the combined Adult Reconstructive Hip and Knee/Musculoskeletal Oncology Fellowship Match did not have accessible web sites. Of the web sites that were accessible, none contained comprehensive information and the majority lacked information that has been previously identified as being important to perspective applicants.

Efficacy of function specific 3D-motifs in enzyme classification according to their EC-numbers.

PubMed

Rahimi, Amir; Madadkar-Sobhani, Armin; Touserkani, Rouzbeh; Goliaei, Bahram

2013-11-07

Due to the increasing number of protein structures with unknown function originated from structural genomics projects, protein function prediction has become an important subject in bioinformatics. Among diverse function prediction methods, exploring known 3D-motifs, which are associated with functional elements in unknown protein structures is one of the most biologically meaningful methods. Homologous enzymes inherit such motifs in their active sites from common ancestors. However, slight differences in the properties of these motifs, results in variation in the reactions and substrates of the enzymes. In this study, we examined the possibility of discriminating highly related active site patterns according to their EC-numbers by 3D-motifs. For each EC-number, the spatial arrangement of an active site, which has minimum average distance to other active sites with the same function, was selected as a representative 3D-motif. In order to characterize the motifs, various points in active site elements were tested. The results demonstrated the possibility of predicting full EC-number of enzymes by 3D-motifs. However, the discriminating power of 3D-motifs varies among different enzyme families and depends on selecting the appropriate points and features. © 2013 Elsevier Ltd. All rights reserved.

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

pocketZebra: a web-server for automated selection and classification of subfamily-specific binding sites by bioinformatic analysis of diverse protein families

PubMed Central

Suplatov, Dmitry; Kirilin, Eugeny; Arbatsky, Mikhail; Takhaveev, Vakil; Švedas, Vytas

2014-01-01

The new web-server pocketZebra implements the power of bioinformatics and geometry-based structural approaches to identify and rank subfamily-specific binding sites in proteins by functional significance, and select particular positions in the structure that determine selective accommodation of ligands. A new scoring function has been developed to annotate binding sites by the presence of the subfamily-specific positions in diverse protein families. pocketZebra web-server has multiple input modes to meet the needs of users with different experience in bioinformatics. The server provides on-site visualization of the results as well as off-line version of the output in annotated text format and as PyMol sessions ready for structural analysis. pocketZebra can be used to study structure–function relationship and regulation in large protein superfamilies, classify functionally important binding sites and annotate proteins with unknown function. The server can be used to engineer ligand-binding sites and allosteric regulation of enzymes, or implemented in a drug discovery process to search for potential molecular targets and novel selective inhibitors/effectors. The server, documentation and examples are freely available at http://biokinet.belozersky.msu.ru/pocketzebra and there are no login requirements. PMID:24852248

Dynamic HypA zinc site is essential for acid viability and proper urease maturation in Helicobacter pylori.

PubMed

Johnson, Ryan C; Hu, Heidi Q; Merrell, D Scott; Maroney, Michael J

2015-04-01

Helicobacter pylori requires urease activity in order to survive in the acid environment of the human stomach. Urease is regulated in part by nickelation, a process that requires the HypA protein, which is a putative nickel metallochaperone that is generally associated with hydrogenase maturation. However, in H. pylori, HypA plays a dual role. In addition to an N-terminal nickel binding site, HypA proteins also contain a structural zinc site that is coordinated by two rigorously conserved CXXC sequences, which in H. pylori are flanked by His residues. These structural Zn sites are known to be dynamic, converting from Zn(Cys)4 centers at pH 7.2 to Zn(Cys)2(His)2 centers at pH 6.3 in the presence of Ni(ii) ions. In this study, mutant strains of H. pylori that express zinc site variants of the HypA protein are used to show that the structural changes in the zinc site are important for the acid viability of the bacterium, and that a reduction in acid viability in these variants can be traced in large measure to deficient urease activity. This in turn leads to a model that connects the Zn(Cys)4 coordination to urease maturation.

Automatic archaeological feature extraction from satellite VHR images

NASA Astrophysics Data System (ADS)

Jahjah, Munzer; Ulivieri, Carlo

2010-05-01

Archaeological applications need a methodological approach on a variable scale able to satisfy the intra-site (excavation) and the inter-site (survey, environmental research). The increased availability of high resolution and micro-scale data has substantially favoured archaeological applications and the consequent use of GIS platforms for reconstruction of archaeological landscapes based on remotely sensed data. Feature extraction of multispectral remotely sensing image is an important task before any further processing. High resolution remote sensing data, especially panchromatic, is an important input for the analysis of various types of image characteristics; it plays an important role in the visual systems for recognition and interpretation of given data. The methods proposed rely on an object-oriented approach based on a theory for the analysis of spatial structures called mathematical morphology. The term "morphology" stems from the fact that it aims at analysing object shapes and forms. It is mathematical in the sense that the analysis is based on the set theory, integral geometry, and lattice algebra. Mathematical morphology has proven to be a powerful image analysis technique; two-dimensional grey tone images are seen as three-dimensional sets by associating each image pixel with an elevation proportional to its intensity level. An object of known shape and size, called the structuring element, is then used to investigate the morphology of the input set. This is achieved by positioning the origin of the structuring element to every possible position of the space and testing, for each position, whether the structuring element either is included or has a nonempty intersection with the studied set. The shape and size of the structuring element must be selected according to the morphology of the searched image structures. Other two feature extraction techniques were used, eCognition and ENVI module SW, in order to compare the results. These techniques were applied to different archaeological sites in Turkmenistan (Nisa) and in Iraq (Babylon); a further change detection analysis was applied to the Babylon site using two HR images as a pre-post second gulf war. We had different results or outputs, taking into consideration the fact that the operative scale of sensed data determines the final result of the elaboration and the output of the information quality, because each of them was sensitive to specific shapes in each input image, we had mapped linear and nonlinear objects, updating archaeological cartography, automatic change detection analysis for the Babylon site. The discussion of these techniques has the objective to provide the archaeological team with new instruments for the orientation and the planning of a remote sensing application.

Catalysis by metal-organic frameworks: fundamentals and opportunities.

PubMed

Ranocchiari, Marco; van Bokhoven, Jeroen Anton

2011-04-14

Crystalline porous materials are extremely important for developing catalytic systems with high scientific and industrial impact. Metal-organic frameworks (MOFs) show unique potential that still has to be fully exploited. This perspective summarizes the properties of MOFs with the aim to understand what are possible approaches to catalysis with these materials. We categorize three classes of MOF catalysts: (1) those with active site on the framework, (2) those with encapsulated active species, and (3) those with active sites attached through post-synthetic modification. We identify the tunable porosity, the ability to fine tune the structure of the active site and its environment, the presence of multiple active sites, and the opportunity to synthesize structures in which key-lock bonding of substrates occurs as the characteristics that distinguish MOFs from other materials. We experience a unique opportunity to imagine and design heterogeneous catalysts, which might catalyze reactions previously thought impossible.

Active site dynamics of ribonuclease.

PubMed Central

Brünger, A T; Brooks, C L; Karplus, M

1985-01-01

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

Engineering Protein Allostery: 1.05 Å Resolution Structure and Enzymatic Properties of a Na[superscript +]-activated Trypsin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, Michael J.; Carrell, Christopher J.; Di Cera, Enrico

2008-05-28

Some trypsin-like proteases are endowed with Na{sup +}-dependent allosteric enhancement of catalytic activity, but this important mechanism has been difficult to engineer in other members of the family. Replacement of 19 amino acids in Streptomyces griseus trypsin targeting the active site and the Na{sup +}-binding site were found necessary to generate efficient Na{sup +} activation. Remarkably, this property was linked to the acquisition of a new substrate selectivity profile similar to that of factor Xa, a Na{sup -} activated protease involved in blood coagulation. The X-ray crystal structure of the mutant trypsin solved to 1.05 {angstrom} resolution defines the engineeredmore » Na{sup +} site and active site loops in unprecedented detail. The results demonstrate that trypsin can be engineered into an efficient allosteric protease, and that Na+ activation is interwoven with substrate selectivity in the trypsin scaffold.« less

Structural Genomics: Correlation Blocks, Population Structure, and Genome Architecture

PubMed Central

Hu, Xin-Sheng; Yeh, Francis C.; Wang, Zhiquan

2011-01-01

An integration of the pattern of genome-wide inter-site associations with evolutionary forces is important for gaining insights into the genomic evolution in natural or artificial populations. Here, we assess the inter-site correlation blocks and their distributions along chromosomes. A correlation block is broadly termed as the DNA segment within which strong correlations exist between genetic diversities at any two sites. We bring together the population genetic structure and the genomic diversity structure that have been independently built on different scales and synthesize the existing theories and methods for characterizing genomic structure at the population level. We discuss how population structure could shape correlation blocks and their patterns within and between populations. Effects of evolutionary forces (selection, migration, genetic drift, and mutation) on the pattern of genome-wide correlation blocks are discussed. In eukaryote organisms, we briefly discuss the associations between the pattern of correlation blocks and genome assembly features in eukaryote organisms, including the impacts of multigene family, the perturbation of transposable elements, and the repetitive nongenic sequences and GC-rich isochores. Our reviews suggest that the observable pattern of correlation blocks can refine our understanding of the ecological and evolutionary processes underlying the genomic evolution at the population level. PMID:21886455

Forest-Site Planning and Prescription for Control of Annosus Root Disease in Ponderosa Pine and Mixed Conifer Stands

Treesearch

John Nesbitt

1989-01-01

In order to successfully combat pathogens such as annosus root rot, the land manager and pathologist must have periodic dialogue about the pest, its identification, effects, impacts, and cures. The author presents four important topics to structure this dialogue. These are (1) training from the pathologist to the silviculturist or other land manager, (2) site specific...

A site-specific approach for assessing the fire risk to structures at the wildland/urban interface

Treesearch

Jack Cohen

1991-01-01

The essence of the wildland/urban interface fire problem is the loss of homes. The problem is not new, but is becoming increasingly important as more homes with inadequate adherence to safety codes are built at the wildland/urban interface. Current regulatory codes are inflexible. Specifications for building and site characteristics cannot be adjusted to accommodate...

Structure, Process, and Culture Differences of Pediatric Trauma Centers Participating in an International Comparative Effectiveness Study of Children with Severe Traumatic Brain Injury.

PubMed

Larsen, Gitte Y; Schober, Michelle; Fabio, Anthony; Wisniewski, Stephen R; Grant, Mary Jo C; Shafi, Nadeem; Bennett, Tellen D; Hirtz, Deborah; Bell, Michael J

2016-06-01

Traumatic brain injury (TBI) is an important worldwide cause of death and disability for children. The Approaches and Decisions for Acute Pediatric TBI (ADAPT) Trial is an observational, cohort study to compare the effectiveness of six aspects of TBI care. Understanding the differences between clinical sites-including their structure, clinical processes, and culture differences-will be necessary to assess differences in outcome from the study and can inform the overall community regarding differences across academic centers. We developed a survey and queried ADAPT site principal investigators with a focus on six domains: (i) hospital, (ii) pediatric intensive care unit (PICU), (iii) medical staff characteristics, (iv) quality of care, (v) medication safety, and (vi) safety culture. Summary statistics were used to describe differences between centers. ADAPT clinical sites that enrolled a subject within the first year (32 US-based, 11 international) were studied. A wide variation in site characteristics was observed in hospital and ICU characteristics, including an almost sevenfold range in ICU size (8-55 beds) and more than fivefold range of overall ICU admissions (537-2623). Nursing staffing (predominantly 1:1 or 1:2) and the presence of pharmacists within the ICU (79 %) were less variable, and most sites "strongly agreed" or "agreed" that Neurosurgery and Critical Care teams worked well together (81.4 %). However, a minority of sites (46 %) used an explicit protocol for treatment of children with severe TBI care. We found a variety of inter-center structure, process, and culture differences. These intrinsic differences between sites may begin to explain why interventional studies have failed to prove efficacy of experimental therapies. Understanding these differences may be an important factor in analyzing future ADAPT trial results and in determining best practices for pediatric severe TBI.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence†

PubMed Central

Martin, Gregory G.; Hostetler, Heather A.; McIntosh, Avery L.; Tichy, Shane E.; Williams, Brad J.; Russell, David H.; Berg, Jeremy M.; Spencer, Thomas A.; Ball, Judith; Kier, Ann B.; Schroeder, Friedhelm

2008-01-01

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2’s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts/caveolae (AF488-CTB), 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488-antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting. PMID:18465878

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Quantitative analysis of American woodcock nest and brood habitat

USGS Publications Warehouse

Bourgeois, A.; Keppie, Daniel M.; Owen, Ray B.

1977-01-01

Sixteen nest and 19 brood sites of American woodcock (Philohela minoI) were examined in northern lower Michigan between 15 April and 15 June 1974 to determine habitat structure associated with these sites. Woodcock hens utilized young, second-growth forest stands which were similar in species composition for both nesting and brood rearing. A multi-varIate discriminant function analysis revealed a significant (P< 0.05) difference, however, in habitat structure. Nest habitat was characterized by lower tree density (2176 trees/ha) and basal area (8.6 m2/ha), by being close to forest openings (7 m) and by being situated on dry, relatively well drained sites. In contrast, woodcock broods were located in sites that had nearly twice the tree density (3934 trees/hal and basal area (16.5 m2/ha), was located over twice as far from forest openings (18 m) and generally occurred on damp sites, near (8 m) standing water. Importance of the habitat features to the species and possible management implications are discussed.

The Crystal Structure of a Cardiovirus RNA-Dependent RNA Polymerase Reveals an Unusual Conformation of the Polymerase Active Site

PubMed Central

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J. M.

2014-01-01

ABSTRACT Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. IMPORTANCE The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses. PMID:24600002

Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations.

PubMed

Correa-Basurto, José; Cuevas-Hernández, Roberto I; Phillips-Farfán, Bryan V; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L; Pérez-González, Óscar A; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G

2015-01-01

Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

PubMed Central

Correa-Basurto, José; Cuevas-Hernández, Roberto I.; Phillips-Farfán, Bryan V.; Martínez-Archundia, Marlet; Romo-Mancillas, Antonio; Ramírez-Salinas, Gema L.; Pérez-González, Óscar A.; Trujillo-Ferrara, José; Mendoza-Torreblanca, Julieta G.

2015-01-01

Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression. PMID:25914622

Best density and structure for uneven-aged northern hardwood management in New England

Treesearch

William B. Leak

2003-01-01

Choice of the best residual density (basal area per acre) and structure (diameter distribution) for uneven-aged management of northern hardwoods is a complex decision that depends on the manager's decision rules, product objectives, site conditions, and - perhaps most important - current stand conditions. In contrast to other recommendations on residual density...

RNase P cleaves transient structures in some riboswitches.

PubMed

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-08-09

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

RNase P cleaves transient structures in some riboswitches

PubMed Central

Altman, Sidney; Wesolowski, Donna; Guerrier-Takada, Cecilia; Li, Yong

2005-01-01

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5′ UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5′ UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested. PMID:16061811

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Y.W.; Robinson, H.; Yeung, N.

2010-05-11

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu hasmore » a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.« less

Roles of Glutamates and Metal ions in a Rationally Designed Nitric Oxide Reductase Based on Myoglobin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y Lin; N Yeung; Y Gao

2011-12-31

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative FeB site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by {approx}110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu hasmore » a different role in redox tuning. More importantly, introducing the second Glu resulted in a {approx}100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the FeB site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more well-defined system to address important chemical and biological issues.« less

Functional and structural characterization of the pentapeptide insertion of Theileria annulata lactate dehydrogenase by site-directed mutagenesis, comparative modeling and molecular dynamics simulations.

PubMed

Erdemir, Aysegul; Mutlu, Ozal

2017-06-01

Lactate dehydrogenase (LDH) is an important metabolic enzyme in glycolysis and it has been considered as the main energy source in many organisms including apicomplexan parasites. Differences at the active site loop of the host and parasite LDH's makes this enzyme an attractive target for drug inhibitors. In this study, five amino acid insertions in the active site pocket of Theileria annulata LDH (TaLDH) were deleted by PCR-based site-directed mutagenesis, expression and activity analysis of mutant and wild type TaLDH enzymes were performed. Removal of the insertion at the active site loop caused production of an inactive enzyme. Furthermore, structures of wild and mutant enzymes were predicted by comparative modeling and the importance of the insertions at the active site loop were also assigned by molecular docking and dynamics simulations in order to evaluate essential role of this loop for the enzymatic activity. Pentapeptide insertion removal resulted in loss of LDH activity due to deletion of Trp96 and conformational change of Arg98 because of loop instability. Analysis of wild type and mutant enzymes with comparative molecular dynamics simulations showed that the fluctuations of the loop residues increase in mutant enzyme. Together with in silico studies, in vitro results revealed that active site loop has a vital role in the enzyme activity and our findings promise hope for the further drug design studies against theileriosis and other apicomplexan parasite diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Anatomy of an engineered NAD-binding site.

PubMed Central

Mittl, P. R.; Berry, A.; Scrutton, N. S.; Perham, R. N.; Schulz, G. E.

1994-01-01

The coenzyme specificity of Escherichia coli glutathione reductase was switched from NADP to NAD by modifying the environment of the 2'-phosphate binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature 343:38-43). In order to analyze the structural changes involved, we have determined 4 high-resolution crystal structures, i.e., the structures of the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD (2.2 A, 16.9%). A comparison of these structures reveals subtle differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a concomitant change of the ribose pucker. The mutations cause a contraction of the local chain fold. Furthermore, the engineered NAD-binding site assumes a less rigid structure than the NADP site of the wild-type enzyme. A superposition of the ligated structures shows a displacement of NAD versus NADP such that the electron pathway from the nicotinamide ring to FAD is elongated, which may explain the lower catalytic efficiency of the mutant. Because the nicotinamide is as much as 15 A from the sites of the mutations, this observation reminds us that mutations may have important long-range consequences that are difficult to anticipate. PMID:7833810

Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

NASA Astrophysics Data System (ADS)

Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

2016-07-01

Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

Structure of a highly acidic β-lactamase from the moderate halophile Chromohalobacter sp. 560 and the discovery of a Cs+-selective binding site

PubMed Central

Arai, Shigeki; Yonezawa, Yasushi; Okazaki, Nobuo; Matsumoto, Fumiko; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide; Tokunaga, Hiroko; Ishibashi, Matsujiro; Blaber, Michael; Tokunaga, Masao; Kuroki, Ryota

2015-01-01

Environmentally friendly absorbents are needed for Sr2+ and Cs+, as the removal of the radioactive Sr2+ and Cs+ that has leaked from the Fukushima Nuclear Power Plant is one of the most important problems in Japan. Halophilic proteins are known to have many acidic residues on their surface that can provide specific binding sites for metal ions such as Cs+ or Sr2+. The crystal structure of a halophilic β-lactamase from Chromohalobacter sp. 560 (HaBLA) was determined to resolutions of between 1.8 and 2.9 Å in space group P31 using X-ray crystallography. Moreover, the locations of bound Sr2+ and Cs+ ions were identified by anomalous X-ray diffraction. The location of one Cs+-specific binding site was identified in HaBLA even in the presence of a ninefold molar excess of Na+ (90 mM Na+/10 mM Cs+). From an activity assay using isothermal titration calorimetry, the bound Sr2+ and Cs+ ions do not significantly affect the enzymatic function of HaBLA. The observation of a selective and high-affinity Cs+-binding site provides important information that is useful for the design of artificial Cs+-binding sites that may be useful in the bioremediation of radioactive isotopes. PMID:25760604

Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification.

PubMed

Cain-Hom, Carol; Splinter, Erik; van Min, Max; Simonis, Marieke; van de Heijning, Monique; Martinez, Maria; Asghari, Vida; Cox, J Colin; Warming, Søren

2017-05-05

Cre/LoxP technology is widely used in the field of mouse genetics for spatial and/or temporal regulation of gene function. For Cre lines generated via pronuclear microinjection of a Cre transgene construct, the integration site is random and in most cases not known. Integration of a transgene can disrupt an endogenous gene, potentially interfering with interpretation of the phenotype. In addition, knowledge of where the transgene is integrated is important for planning of crosses between animals carrying a conditional allele and a given Cre allele in case the alleles are on the same chromosome. We have used targeted locus amplification (TLA) to efficiently map the transgene location in seven previously published Cre and CreERT2 transgenic lines. In all lines, transgene insertion was associated with structural changes of variable complexity, illustrating the importance of testing for rearrangements around the integration site. In all seven lines the exact integration site and breakpoint sequences were identified. Our methods, data and genotyping assays can be used as a resource for the mouse community and our results illustrate the power of the TLA method to not only efficiently map the integration site of any transgene, but also provide additional information regarding the transgene integration events. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structural basis of carbohydrate recognition by lectin II from Ulex europaeus, a protein with a promiscuous carbohydrate-binding site.

PubMed

Loris, R; De Greve, H; Dao-Thi, M H; Messens, J; Imberty, A; Wyns, L

2000-08-25

Protein-carbohydrate interactions are the language of choice for inter- cellular communication. The legume lectins form a large family of homologous proteins that exhibit a wide variety of carbohydrate specificities. The legume lectin family is therefore highly suitable as a model system to study the structural principles of protein-carbohydrate recognition. Until now, structural data are only available for two specificity families: Man/Glc and Gal/GalNAc. No structural data are available for any of the fucose or chitobiose specific lectins. The crystal structure of Ulex europaeus (UEA-II) is the first of a legume lectin belonging to the chitobiose specificity group. The complexes with N-acetylglucosamine, galactose and fucosylgalactose show a promiscuous primary binding site capable of accommodating both N-acetylglucos amine or galactose in the primary binding site. The hydrogen bonding network in these complexes can be considered suboptimal, in agreement with the low affinities of these sugars. In the complexes with chitobiose, lactose and fucosyllactose this suboptimal hydrogen bonding network is compensated by extensive hydrophobic interactions in a Glc/GlcNAc binding subsite. UEA-II thus forms the first example of a legume lectin with a promiscuous binding site and illustrates the importance of hydrophobic interactions in protein-carbohydrate complexes. Together with other known legume lectin crystal structures, it shows how different specificities can be grafted upon a conserved structural framework. Copyright 2000 Academic Press.

Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

PubMed

Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

2011-07-01

The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases.

Recognition of functional sites in protein structures.

PubMed

Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

2004-06-04

Recognition of regions on the surface of one protein, that are similar to a binding site of another is crucial for the prediction of molecular interactions and for functional classifications. We first describe a novel method, SiteEngine, that assumes no sequence or fold similarities and is able to recognize proteins that have similar binding sites and may perform similar functions. We achieve high efficiency and speed by introducing a low-resolution surface representation via chemically important surface points, by hashing triangles of physico-chemical properties and by application of hierarchical scoring schemes for a thorough exploration of global and local similarities. We proceed to rigorously apply this method to functional site recognition in three possible ways: first, we search a given functional site on a large set of complete protein structures. Second, a potential functional site on a protein of interest is compared with known binding sites, to recognize similar features. Third, a complete protein structure is searched for the presence of an a priori unknown functional site, similar to known sites. Our method is robust and efficient enough to allow computationally demanding applications such as the first and the third. From the biological standpoint, the first application may identify secondary binding sites of drugs that may lead to side-effects. The third application finds new potential sites on the protein that may provide targets for drug design. Each of the three applications may aid in assigning a function and in classification of binding patterns. We highlight the advantages and disadvantages of each type of search, provide examples of large-scale searches of the entire Protein Data Base and make functional predictions.

Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

Three-dimensional crystal structure of recombinant murine interferon-beta.

PubMed Central

Senda, T; Shimazu, T; Matsuda, S; Kawano, G; Shimizu, H; Nakamura, K T; Mitsui, Y

1992-01-01

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices. Images PMID:1505514

Binding Leverage as a Molecular Basis for Allosteric Regulation

PubMed Central

Mitternacht, Simon; Berezovsky, Igor N.

2011-01-01

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. PMID:21935347

XenoSite server: a web-available site of metabolism prediction tool.

PubMed

Matlock, Matthew K; Hughes, Tyler B; Swamidass, S Joshua

2015-04-01

Cytochrome P450 enzymes (P450s) are metabolic enzymes that process the majority of FDA-approved, small-molecule drugs. Understanding how these enzymes modify molecule structure is key to the development of safe, effective drugs. XenoSite server is an online implementation of the XenoSite, a recently published computational model for P450 metabolism. XenoSite predicts which atomic sites of a molecule--sites of metabolism (SOMs)--are modified by P450s. XenoSite server accepts input in common chemical file formats including SDF and SMILES and provides tools for visualizing the likelihood that each atomic site is a site of metabolism for a variety of important P450s, as well as a flat file download of SOM predictions. XenoSite server is available at http://swami.wustl.edu/xenosite. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Hydrogeological controls on spatial patterns of groundwater discharge in peatlands

NASA Astrophysics Data System (ADS)

Hare, Danielle K.; Boutt, David F.; Clement, William P.; Hatch, Christine E.; Davenport, Glorianna; Hackman, Alex

2017-11-01

Peatland environments provide important ecosystem services including water and carbon storage, nutrient processing and retention, and wildlife habitat. However, these systems and the services they provide have been degraded through historical anthropogenic agricultural conversion and dewatering practices. Effective wetland restoration requires incorporating site hydrology and understanding groundwater discharge spatial patterns. Groundwater discharge maintains wetland ecosystems by providing relatively stable hydrologic conditions, nutrient inputs, and thermal buffering important for ecological structure and function; however, a comprehensive site-specific evaluation is rarely feasible for such resource-constrained projects. An improved process-based understanding of groundwater discharge in peatlands may help guide ecological restoration design without the need for invasive methodologies and detailed site-specific investigation. Here we examine a kettle-hole peatland in southeast Massachusetts historically modified for commercial cranberry farming. During the time of our investigation, a large process-based ecological restoration project was in the assessment and design phases. To gain insight into the drivers of site hydrology, we evaluated the spatial patterning of groundwater discharge and the subsurface structure of the peatland complex using heat-tracing methods and ground-penetrating radar. Our results illustrate that two groundwater discharge processes contribute to the peatland hydrologic system: diffuse lower-flux marginal matrix seepage and discrete higher-flux preferential-flow-path seepage. Both types of groundwater discharge develop through interactions with subsurface peatland basin structure, often where the basin slope is at a high angle to the regional groundwater gradient. These field observations indicate strong correlation between subsurface structures and surficial groundwater discharge. Understanding these general patterns may allow resource managers to more efficiently predict and locate groundwater seepage, confirm these using remote sensing technologies, and incorporate this information into restoration design for these critical ecosystems.

Long-term spatiotemporal stability and dynamic changes in helminth infracommunities of bank voles (Myodes glareolus) in NE Poland.

PubMed

Grzybek, Maciej; Bajer, Anna; Bednarska, Małgorzata; Al-Sarraf, Mohammed; Behnke-Borowczyk, Jolanta; Harris, Philip D; Price, Stephen J; Brown, Gabrielle S; Osborne, Sarah-Jane; Siński, Edward; Behnke, Jerzy M

2015-12-01

Parasites are considered to be an important selective force in host evolution but ecological studies of host-parasite systems are usually short-term providing only snap-shots of what may be dynamic systems. We have conducted four surveys of helminths of bank voles at three ecologically similar woodland sites in NE Poland, spaced over a period of 11 years, to assess the relative importance of temporal and spatial effects on helminth infracommunities. Some measures of infracommunity structure maintained relative stability: the rank order of prevalence and abundance of Heligmosomum mixtum, Heligmosomoides glareoli and Mastophorus muris changed little between the four surveys. Other measures changed markedly: dynamic changes were evident in Syphacia petrusewiczi which declined to local extinction, while the capillariid Aonchotheca annulosa first appeared in 2002 and then increased in prevalence and abundance over the remaining three surveys. Some species are therefore dynamic and both introductions and extinctions can be expected in ecological time. At higher taxonomic levels and for derived measures, year and host-age effects and their interactions with site are important. Our surveys emphasize that the site of capture is the major determinant of the species contributing to helminth community structure, providing some predictability in these systems.

Evaluation of normal findings using a detailed and focused technique for transcutaneous abdominal ultrasonography in the horse

PubMed Central

2014-01-01

Background Ultrasonography is an important diagnostic tool in the investigation of abdominal disease in the horse. Several factors may affect the ability to image different structures within the abdomen. The aim of the study was to describe the repeatability of identification of abdominal structures in normal horses using a detailed ultrasonographic examination technique and using a focused, limited preparation technique. Methods A detailed abdominal ultrasound examination was performed in five normal horses, repeated on five occasions (total of 25 examinations). The abdomen was divided into ten different imaging sites, and structures identified in each site were recorded. Five imaging sites were then selected for a single focused ultrasound examination in 20 normal horses. Limited patient preparation was performed. Structures were recorded as ‘identified’ if ultrasonographic features could be distinguished. The location of organs and their frequency of identification were recorded. Data from both phases were analysed to determine repeatability of identification of structures in each examination (irrespective of imaging site), and for each imaging site. Results Caecum, colon, spleen, liver and right kidney were repeatably identified using the detailed technique, and had defined locations. Large colon and right kidney were identified in 100% of examinations with both techniques. Liver, spleen, caecum, duodenum and other small intestine were identified more frequently with the detailed examination. Small intestine was most frequently identified in the ventral abdomen, its identification varied markedly within and between horses, and required repeated examinations in some horses. Left kidney could not be identified in every horse using either technique. Sacculated colon was identified in all ventral sites, and was infrequently identified in dorsal sites. Conclusions Caecum, sacculated large intestine, spleen, liver and right kidney were consistently identified with both techniques. There were some normal variations which should be considered when interpreting ultrasonographic findings in clinical cases: left kidney was not always identified, sacculated colon was occasionally identified in dorsal flank sites. Multiple imaging sites and repeated examinations may be required to identify small intestine. A focused examination identified most key structures, but has some limitations compared to a detailed examination. PMID:25238559

Amphoteric doping of praseodymium Pr 3+ in SrTiO 3 grain boundaries

DOE PAGES

Yang, H.; Lee, H. S.; Kotula, P. G.; ...

2015-03-26

Charge Compensation in rare-earth Praseodymium (Pr 3+) doped SrTiO 3 plays an important role in determining the overall photoluminescence properties of the system. Here, the Pr 3+ doping behavior in SrTiO 3 grain boundaries (GBs) is analyzed using aberration corrected scanning transmission electron microscopy (STEM). The presence of Pr 3+ induces structure variations and changes the statistical prevalence of GB structures. In contrast to the assumption that Pr 3+ substitutes for A site as expected in the bulk, Pr 3+ is found to substitute both Sr and Ti sites inside GBs with the highest concentration in the Ti sites. Asmore » a result, this amphoteric doping behavior in the boundary plane is further confirmed by first principles theoretical calculations.« less

Amphoteric Doping of Praseodymium Pr3+ in SrTiO3 Grain Boundaries

DOE PAGES

Yang, Hao; Lee, H. S.; Kotula, Paul G.; ...

2015-03-23

Charge Compensation in rare-earth Praseodymium (Pr 3+) doped SrTiO 3 plays an important role in determining the overall photoluminescence properties of the system. Here, the Pr 3+ doping behavior in SrTiO 3 grain boundaries (GBs) is analyzed using aberration corrected scanning transmission electron microscopy (STEM). The presence of Pr 3+ induces structure variations and changes the statistical prevalence of GB structures. In contrast to the assumption that Pr 3+ substitutes for A site as expected in the bulk, Pr 3+ is found to substitute both Sr and Ti sites inside GBs with the highest concentration in the Ti sites. Asmore » a result, this amphoteric doping behavior in the boundary plane is further confirmed by first principles theoretical calculations.« less

Site Transfer Functions of Three-Component Ground Motion in Western Turkey

NASA Astrophysics Data System (ADS)

Ozgur Kurtulmus, Tevfik; Akyol, Nihal; Camyildiz, Murat; Gungor, Talip

2015-04-01

Because of high seismicity accommodating crustal deformation and deep graben structures, on which have, urbanized and industrialized large cities in western Turkey, the importance of site-specific seismic hazard assessments becomes more crucial. Characterizing source, site and path effects is important for both assessing the seismic hazard in a specific region and generation of the building codes/or renewing previous ones. In this study, we evaluated three-component recordings for micro- and moderate-size earthquakes with local magnitudes ranging between 2.0 and 5.6. This dataset is used for site transfer function estimations, utilizing two different spectral ratio approaches 'Standard Spectral Ratio-(SSR)' and 'Horizontal to Vertical Spectral Ratio-(HVSR)' and a 'Generalized Inversion Technique-(GIT)' to highlight site-specific seismic hazard potential of deep basin structures of the region. Obtained transfer functions revealed that the sites located near the basin edges are characterized by broader HVSR curves. Broad HVSR peaks could be attributed to the complexity of wave propagation related to significant 2D/3D velocity variations at the sediment-bedrock interface near the basin edges. Comparison of HVSR and SSR estimates for the sites located on the grabens showed that SSR estimates give larger values at lower frequencies which could be attributed to lateral variations in regional velocity and attenuation values caused by basin geometry and edge effects. However, large amplitude values of vertical component GIT site transfer functions were observed at varying frequency ranges for some of the stations. These results imply that vertical component of ground motion is not amplification free. Contamination of HVSR site transfer function estimates at different frequency bands could be related to complexities in the wave field caused by deep or shallow heterogeneities in the region such as differences in the basin geometries, fracturing and fluid saturation along different propagation paths. The results also show that, even if the site is located on a horst, the presence of weathered zones near the surface could cause moderate frequency dependent site effects.

Electrochemical and Density Functional Theory Study on the Reactivity of Fisetin and Its Radicals: Implications on in Vitro Antioxidant Activity

NASA Astrophysics Data System (ADS)

Marković, Zoran S.; Mentus, Slavko V.; Dimitrić Marković, Jasmina M.

2009-12-01

Antioxidative properties of naturally occurring flavon-3-ol, fisetin, were examined by both cyclic voltammetry and quantum-chemical based calculations. The three voltametrically detectable consecutive steps, reflected the fisetin molecular structure, catecholic structural unit in the ring B, C3-OH, and C7-OH groups in the rings C and A. Oxidation potential values, used as quantitative parameter in determining its oxidation capability, indicated good antioxidative properties found with this molecule. Oxidation of the C3'C4' dixydroxy moiety at the B ring occurred first at the lowest positive potentials. The first oxidation step induced fast intramolecular transformations in which the C3 hydroxy group disappeared and the product of this transformation participated in the second oxidation step. The highest potential of oxidation was attributed to the oxidation of C7 hydroxy group. The structural and electronic features of fisetin were investigated at the B3LYP/6-311++G** level of theory. Particularly, the interest was focused on the C3' and C4'-OH sites in the B ring and on C3-OH site in the C ring. The calculated bond dissociation enthalpy values for all OH sites of fisetin clearly indicated the importance of the B ring and C3' and C4'-OH group. The importance of keto-enol tautomerism has also been considered. The analysis also included the Mulliken spin density distribution for the radicals formed after H removal on each OH site. The results showed the higher values of the BDE on the C7-OH and C3-OH sites.

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W

PubMed Central

Barleben, Leif; Panjikar, Santosh; Ruppert, Martin; Koepke, Juergen; Stöckigt, Joachim

2007-01-01

Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG's residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases. PMID:17890378

A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by Its dodecameric structure

DOE PAGES

Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy; ...

2015-01-23

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence ofmore » a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis.« less

A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by its dodecameric structure.

PubMed

Filippova, Ekaterina V; Kuhn, Misty L; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Ballicora, Miguel A; Anderson, Wayne F

2015-03-27

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis. Copyright © 2015. Published by Elsevier Ltd.

A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by Its dodecameric structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Filippova, Ekaterina V.; Kuhn, Misty L.; Osipiuk, Jerzy

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence ofmore » a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis.« less

Community structure and diversity of tropical forest mammals: data from a global camera trap network.

PubMed

Ahumada, Jorge A; Silva, Carlos E F; Gajapersad, Krisna; Hallam, Chris; Hurtado, Johanna; Martin, Emanuel; McWilliam, Alex; Mugerwa, Badru; O'Brien, Tim; Rovero, Francesco; Sheil, Douglas; Spironello, Wilson R; Winarni, Nurul; Andelman, Sandy J

2011-09-27

Terrestrial mammals are a key component of tropical forest communities as indicators of ecosystem health and providers of important ecosystem services. However, there is little quantitative information about how they change with local, regional and global threats. In this paper, the first standardized pantropical forest terrestrial mammal community study, we examine several aspects of terrestrial mammal species and community diversity (species richness, species diversity, evenness, dominance, functional diversity and community structure) at seven sites around the globe using a single standardized camera trapping methodology approach. The sites-located in Uganda, Tanzania, Indonesia, Lao PDR, Suriname, Brazil and Costa Rica-are surrounded by different landscape configurations, from continuous forests to highly fragmented forests. We obtained more than 51 000 images and detected 105 species of mammals with a total sampling effort of 12 687 camera trap days. We find that mammal communities from highly fragmented sites have lower species richness, species diversity, functional diversity and higher dominance when compared with sites in partially fragmented and continuous forest. We emphasize the importance of standardized camera trapping approaches for obtaining baselines for monitoring forest mammal communities so as to adequately understand the effect of global, regional and local threats and appropriately inform conservation actions.

Multi-scale habitat selection in highly territorial bird species: Exploring the contribution of nest, territory and landscape levels to site choice in breeding rallids (Aves: Rallidae)

NASA Astrophysics Data System (ADS)

Jedlikowski, Jan; Chibowski, Piotr; Karasek, Tomasz; Brambilla, Mattia

2016-05-01

Habitat selection often involves choices made at different spatial scales, but the underlying mechanisms are still poorly understood, and studies that investigate the relative importance of individual scales are rare. We investigated the effect of three spatial scales (landscape, territory, nest-site) on the occurrence pattern of little crake Zapornia parva and water rail Rallus aquaticus at 74 ponds in the Masurian Lakeland, Poland. Habitat structure, food abundance and water chemical parameters were measured at nests and random points within landscape plots (from 300-m to 50-m radius), territory (14-m) and nest-site plots (3-m). Regression analyses suggested that the most relevant scale was territory level, followed by landscape, and finally by nest-site for both species. Variation partitioning confirmed this pattern for water rail, but also highlighted the importance of nest-site (the level explaining the highest share of unique variation) for little crake. The most important variables determining the occurrence of both species were water body fragmentation (landscape), vegetation density (territory) and water depth (at territory level for little crake, and at nest-site level for water rail). Finally, for both species multi-scale models including factors from different levels were more parsimonious than single-scale ones, i.e. habitat selection was likely a multi-scale process. The importance of particular spatial scales seemed more related to life-history traits than to the extent of the scales considered. In the case of our study species, the territory level was highly important likely because both rallids have to obtain all the resources they need (nest site, food and mates) in relatively small areas, the multi-purpose territories they defend.

Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

PubMed Central

Kaiserman, Dion; Buckle, Ashley M.; Van Damme, Petra; Irving, James A.; Law, Ruby H. P.; Matthews, Antony Y.; Bashtannyk-Puhalovich, Tanya; Langendorf, Chris; Thompson, Philip; Vandekerckhove, Joël; Gevaert, Kris; Whisstock, James C.; Bird, Phillip I.

2009-01-01

Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-Å X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the active-site serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu–Glu motif at positions 192–193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-Å structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone. PMID:19299505

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.

PubMed

Repanas, Kostas; Zingler, Nora; Layer, Liliana E; Schumann, Gerald G; Perrakis, Anastassis; Weichenrieder, Oliver

2007-01-01

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for approximately 28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE PAGES

Thal, David M.; Sun, Bingfa; Feng, Dan; ...

2016-03-09

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Crystal structures of the M 1 and M 4 muscarinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thal, David M.; Sun, Bingfa; Feng, Dan

Muscarinic M1–M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer’s disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. In this paper, we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 andmore » M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. Finally, we also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.« less

Structure and DNA-binding of meiosis-specific protein Hop2

NASA Astrophysics Data System (ADS)

Zhou, Donghua; Moktan, Hem; Pezza, Roberto

2014-03-01

Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 (Hop2), which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood. As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 (1-84) using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment. Information of these sites was used to guide protein-DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 (connecting strands 2 and 3) transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.

Assessment of ligand binding at a site relevant to SOD1 oxidation and aggregation.

PubMed

Manjula, Ramu; Wright, Gareth S A; Strange, Richard W; Padmanabhan, Balasundaram

2018-05-01

Cu/Zn superoxide dismutase-1 (SOD1) mutations are causative for a subset of amyotrophic lateral sclerosis (ALS) cases. These mutations lead to structural instability, aggregation and ultimately motor neuron death. We have determined crystal structures of SOD1 in complex with a naphthalene-catechol-linked compound which binds with low micro-molar affinity to a site important for oxidative damage-induced aggregation. SOD1 Trp32 oxidation is indeed significantly inhibited by ligand binding. Our work shows how compound linking can be applied successfully to ligand interactions on the SOD1 surface to generate relatively good binding strength. The ligand, positioned in a region important for SOD1 fibrillation, offers the possibility that it, or a similar compound, could prevent the abnormal self-association that drives SOD1 toxicity in ALS. © 2018 Federation of European Biochemical Societies.

Considering Spatial Scale and Reproductive Consequences of Habitat Selection when Managing Grasslands for a Threatened Species

PubMed Central

Pearson, Scott F.; Knapp, Shannon M.

2016-01-01

Habitat selection that has fitness consequences has important implications for conservation activities. For example, habitat characteristics that influence nest success in birds can be manipulated to improve habitat quality with the goal of ultimately improving reproductive success. We examined habitat selection by the threatened streaked horned lark (Eremophila alpestris strigata) at both the breeding-site (territory) and nest-site scales. Larks were selective at both spatial scales but with contrasting selection. At the territory scale, male larks selected sparsely vegetated grasslands with relatively short vegetation. At the nest-site scale, female larks selected sites within territories with higher vegetation density and more perennial forbs. These nest-site scale choices had reproductive consequences, with greater nest success in areas with higher densities of perennial forbs. We experimentally manipulated lark habitat structure in an attempt to mimic the habitat conditions selected by larks by using late summer prescribed fires. After the burn, changes in vegetation structure were in the direction preferred by larks but habitat effects attenuated by the following year. Our results highlight the importance of evaluating habitat selection at spatial scales appropriate to the species of interest, especially when attempting to improve habitat quality for rare and declining species. They also highlight the importance of conducting restoration activities in a research context. For example, because the sparsely vegetated conditions created by fire attenuate, there may be value in examining more frequent burns or hotter fires as the next management and research action. We hope the design outlined in this study will serve as an integrated research and management example for conserving grassland birds generally. PMID:27322196

Scale-Dependence of Processes Structuring Dung Beetle Metacommunities Using Functional Diversity and Community Deconstruction Approaches

PubMed Central

da Silva, Pedro Giovâni; Hernández, Malva Isabel Medina

2015-01-01

Community structure is driven by mechanisms linked to environmental, spatial and temporal processes, which have been successfully addressed using metacommunity framework. The relative importance of processes shaping community structure can be identified using several different approaches. Two approaches that are increasingly being used are functional diversity and community deconstruction. Functional diversity is measured using various indices that incorporate distinct community attributes. Community deconstruction is a way to disentangle species responses to ecological processes by grouping species with similar traits. We used these two approaches to determine whether they are improvements over traditional measures (e.g., species composition, abundance, biomass) for identification of the main processes driving dung beetle (Scarabaeinae) community structure in a fragmented mainland-island landscape in southern Brazilian Atlantic Forest. We sampled five sites in each of four large forest areas, two on the mainland and two on the island. Sampling was performed in 2012 and 2013. We collected abundance and biomass data from 100 sampling points distributed over 20 sampling sites. We studied environmental, spatial and temporal effects on dung beetle community across three spatial scales, i.e., between sites, between areas and mainland-island. The γ-diversity based on species abundance was mainly attributed to β-diversity as a consequence of the increase in mean α- and β-diversity between areas. Variation partitioning on abundance, biomass and functional diversity showed scale-dependence of processes structuring dung beetle metacommunities. We identified two major groups of responses among 17 functional groups. In general, environmental filters were important at both local and regional scales. Spatial factors were important at the intermediate scale. Our study supports the notion of scale-dependence of environmental, spatial and temporal processes in the distribution and functional organization of Scarabaeinae beetles. We conclude that functional diversity may be used as a complementary approach to traditional measures, and that community deconstruction allows sufficient disentangling of responses of different trait-based groups. PMID:25822150

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

PubMed Central

Jagtap, Pravin Kumar Ankush; Soni, Vijay; Vithani, Neha; Jhingan, Gagan Deep; Bais, Vaibhav Singh; Nandicoori, Vinay Kumar; Prakash, Balaji

2012-01-01

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmUMtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmUMtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmUMtb. In this SN2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmUMtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmUMtb, which may be exploited for the development of inhibitors specific to GlmU. PMID:22969087

Drivers of cyanobacterial diversity and community composition in mangrove soils in south-east Brazil.

PubMed

Rigonato, Janaina; Kent, Angela D; Alvarenga, Danillo O; Andreote, Fernando D; Beirigo, Raphael M; Vidal-Torrado, Pablo; Fiore, Marli F

2013-04-01

Cyanobacteria act as primary producers of carbon and nitrogen in nutrient-poor ecosystems such as mangroves. This important group of microorganisms plays a critical role in sustaining the productivity of mangrove ecosystems, but the structure and function of cyanobacteria assemblages can be perturbed by anthropogenic influences. The aim of this work was to assess the community structure and ecological drivers that influence the cyanobacterial community harboured in two Brazilian mangrove soils, and examine the long-term effects of oil contamination on these keystone species. Community fingerprinting results showed that, although cyanobacterial communities are distinct between the two mangroves, the structure and diversity of the assemblages exhibit similar responses to environmental gradients. In each ecosystem, cyanobacteria occupying near-shore areas were similar in composition, indicating importance of marine influences for structuring the community. Analysis of 16S rRNA sequences revealed the presence of diverse cyanobacterial communities in mangrove sediments, with clear differences among mangrove habitats along a transect from shore to forest. While near-shore sites in both mangroves were mainly occupied by Prochlorococcus and Synechococcus genera, sequences retrieved from other mangrove niches were mainly affiliated with uncultured cyanobacterial 16S rRNA. The most intriguing finding was the large number of potentially novel cyanobacteria 16S rRNA sequences obtained from a previously oil-contaminated site. The abundance of cyanobacterial 16S rRNA sequences observed in sites with a history of oil contamination was significantly lower than in the unimpacted areas. This study emphasized the role of environmental drivers in determining the structure of cyanobacterial communities in mangrove soils, and suggests that anthropogenic impacts may also act as ecological filters that select cyanobacterial taxa. These results are an important contribution to our understanding of the composition and relative abundance of previously poorly described cyanobacterial assemblages in mangrove ecosystems. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Local structure investigation of Ga and Yb dopants in Co 4 Sb 12 skutterudites

DOE PAGES

Hu, Yanyun; Chen, Ning; Clancy, J. P.; ...

2017-12-29

We report our x-ray absorption spectroscopy studies at both Ga K-edge and Yb L 2-edge to elucidate the local structure of Ga and Yb dopants in Yb xGa yCo 4Sb 12. Our extended x-ray absorption fine structure (EXAFS) data confirm that Ga atoms occupy two crystallographic sites: one is the 24g site replacing Sb, and the other is the 2a site in the off-center void position. We find that the occupancy ratio of these two sites varies significantly as a function of the filling fraction of additional Yb, which exclusively occupies the 2a on-center site. At low concentrations of Yb,more » Ga 24g and Ga 2a dopants coexist and they form a charge-compensated compound defect proposed by Qiu et al. [Adv. Mater. 23, 3194 (2013)]. The Ga 24g occupancy increases gradually with increasing Yb concentration, and almost all Ga occupies the 24g site for the highest Yb concentration (x = 0.4). In addition to the local crystal structure evidence provided by our EXAFS data, we also present x-ray absorption near-edge structure (XANES) spectra, which show a small Ga K-edge energy shift as a function of Yb concentration consistent with the change from predominantly Ga 2a to Ga 24g states. Our result suggests that the increased solubility of Yb in Yb-Ga co-doped Co 4Sb 12 skutterudites is due to the increased Ga 24g electron acceptor, and thus provides an important strategy to optimize the carrier concentration in partially filled skutterudites.« less

Local structure investigation of Ga and Yb dopants in Co4Sb12 skutterudites

NASA Astrophysics Data System (ADS)

Hu, Yanyun; Chen, Ning; Clancy, J. P.; Salvador, James R.; Kim, Chang-Yong; Shi, Xiaoya; Li, Qiang; Kim, Young-June

2017-12-01

We report comprehensive x-ray absorption spectroscopy studies at both the Ga K edge and Yb L2 edge to elucidate the local structure of Ga and Yb dopants in YbxGayCo4Sb12 . Our extended x-ray absorption fine structure (EXAFS) data confirm that Ga atoms occupy two crystallographic sites: one is the 24 g site replacing Sb, and the other is the 2 a site in the off-center void position. We find that the occupancy ratio of these two sites varies significantly as a function of the filling fraction of additional Yb, which exclusively occupies the 2 a on-center site. At low concentrations of Yb, Ga24 g and Ga2 a dopants coexist and they form a charge-compensated compound defect proposed by Qiu et al. [Adv. Funct. Mater. 23, 3194 (2013), 10.1002/adfm.201202571]. The Ga24 g occupancy increases gradually with increasing Yb concentration, and almost all Ga occupies the 24 g site for the highest Yb concentration studied (x =0.4 ). In addition to the local structural evidence provided by our EXAFS data, we also present x-ray absorption near-edge structure (XANES) spectra, which show a small Ga K -edge energy shift as a function of Yb concentration consistent with the change from predominantly Ga2 a to Ga24 g states. Our result suggests that the increased solubility of Yb in Yb-Ga co-doped Co4Sb12 skutterudites is due to the increased Ga24 g electron acceptor, and thus provides an important strategy to optimize the carrier concentration in partially filled skutterudites.

Local structure investigation of Ga and Yb dopants in Co 4 Sb 12 skutterudites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Yanyun; Chen, Ning; Clancy, J. P.

We report our x-ray absorption spectroscopy studies at both Ga K-edge and Yb L 2-edge to elucidate the local structure of Ga and Yb dopants in Yb xGa yCo 4Sb 12. Our extended x-ray absorption fine structure (EXAFS) data confirm that Ga atoms occupy two crystallographic sites: one is the 24g site replacing Sb, and the other is the 2a site in the off-center void position. We find that the occupancy ratio of these two sites varies significantly as a function of the filling fraction of additional Yb, which exclusively occupies the 2a on-center site. At low concentrations of Yb,more » Ga 24g and Ga 2a dopants coexist and they form a charge-compensated compound defect proposed by Qiu et al. [Adv. Mater. 23, 3194 (2013)]. The Ga 24g occupancy increases gradually with increasing Yb concentration, and almost all Ga occupies the 24g site for the highest Yb concentration (x = 0.4). In addition to the local crystal structure evidence provided by our EXAFS data, we also present x-ray absorption near-edge structure (XANES) spectra, which show a small Ga K-edge energy shift as a function of Yb concentration consistent with the change from predominantly Ga 2a to Ga 24g states. Our result suggests that the increased solubility of Yb in Yb-Ga co-doped Co 4Sb 12 skutterudites is due to the increased Ga 24g electron acceptor, and thus provides an important strategy to optimize the carrier concentration in partially filled skutterudites.« less

Metal stabilization of collagen and de novo designed mimetic peptides

PubMed Central

Parmar, Avanish S.; Xu, Fei; Pike, Douglas H.; Belure, Sandeep V.; Hasan, Nida F.; Drzewiecki, Kathryn E.; Shreiber, David I.; Nanda, Vikas

2017-01-01

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix. PMID:26225466

Metal Stabilization of Collagen and de Novo Designed Mimetic Peptides.

PubMed

Parmar, Avanish S; Xu, Fei; Pike, Douglas H; Belure, Sandeep V; Hasan, Nida F; Drzewiecki, Kathryn E; Shreiber, David I; Nanda, Vikas

2015-08-18

We explore the design of metal binding sites to modulate triple-helix stability of collagen and collagen-mimetic peptides. Globular proteins commonly utilize metals to connect tertiary structural elements that are well separated in sequence, constraining structure and enhancing stability. It is more challenging to engineer structural metals into fibrous protein scaffolds, which lack the extensive tertiary contacts seen in globular proteins. In the collagen triple helix, the structural adjacency of the carboxy-termini of the three chains makes this region an attractive target for introducing metal binding sites. We engineered His3 sites based on structural modeling constraints into a series of designed homotrimeric and heterotrimeric peptides, assessing the capacity of metal binding to improve stability and in the case of heterotrimers, affect specificity of assembly. Notable enhancements in stability for both homo- and heteromeric systems were observed upon addition of zinc(II) and several other metal ions only when all three histidine ligands were present. Metal binding affinities were consistent with the expected Irving-Williams series for imidazole. Unlike other metals tested, copper(II) also bound to peptides lacking histidine ligands. Acetylation of the peptide N-termini prevented copper binding, indicating proline backbone amide metal-coordination at this site. Copper similarly stabilized animal extracted Type I collagen in a metal-specific fashion, highlighting the potential importance of metal homeostasis within the extracellular matrix.

Structure-Activity Relationship and Molecular Mechanics Reveal the Importance of Ring Entropy in the Biosynthesis and Activity of a Natural Product.

PubMed

Tran, Hai L; Lexa, Katrina W; Julien, Olivier; Young, Travis S; Walsh, Christopher T; Jacobson, Matthew P; Wells, James A

2017-02-22

Macrocycles are appealing drug candidates due to their high affinity, specificity, and favorable pharmacological properties. In this study, we explored the effects of chemical modifications to a natural product macrocycle upon its activity, 3D geometry, and conformational entropy. We chose thiocillin as a model system, a thiopeptide in the ribosomally encoded family of natural products that exhibits potent antimicrobial effects against Gram-positive bacteria. Since thiocillin is derived from a genetically encoded peptide scaffold, site-directed mutagenesis allows for rapid generation of analogues. To understand thiocillin's structure-activity relationship, we generated a site-saturation mutagenesis library covering each position along thiocillin's macrocyclic ring. We report the identification of eight unique compounds more potent than wild-type thiocillin, the best having an 8-fold improvement in potency. Computational modeling of thiocillin's macrocyclic structure revealed a striking requirement for a low-entropy macrocycle for activity. The populated ensembles of the active mutants showed a rigid structure with few adoptable conformations while inactive mutants showed a more flexible macrocycle which is unfavorable for binding. This finding highlights the importance of macrocyclization in combination with rigidifying post-translational modifications to achieve high-potency binding.

Structural-electrochemical relations in the aqueous copper hexacyanoferrate-zinc system examined by synchrotron X-ray diffraction

NASA Astrophysics Data System (ADS)

Renman, Viktor; Ojwang, Dickson O.; Valvo, Mario; Gómez, Cesar Pay; Gustafsson, Torbjörn; Svensson, Gunnar

2017-11-01

The storage process of Zn2+ in the Prussian blue analogue (PBA) copper hexacyanoferrate (Cu[Fe(CN)6]2/3·nH2O - CuHCF) framework structure in a context of rechargeable aqueous batteries is examined by means of in operando synchrotron X-ray diffraction. Via sequential unit-cell parameter refinements of time-resolved diffraction data, it is revealed that the step-profile of the cell output voltage curves during repeated electrochemical insertion and removal of Zn2+ in the CuHCF host structure is associated with a non-linear contraction and expansion of the unit-cell in the range 0.36 < x < 1.32 for Znx/3Cu[Fe(CN)6]2/3·nH2O. For a high insertion cation content there is no apparent change in the unit-cell contraction. Furthermore, a structural analysis with respect to the occupancies of possible Zn2+ sites suggests that the Fe(CN)6 vacancies within the CuHCF framework play an important role in the structural-electrochemical behavior of this particular system. More specifically, it is observed that Zn2+ swaps position during electrochemical cycling, hopping between cavity sites to vacant ferricyanide sites.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR

NASA Astrophysics Data System (ADS)

Lange, Adam; Giller, Karin; Hornig, Sönke; Martin-Eauclaire, Marie-France; Pongs, Olaf; Becker, Stefan; Baldus, Marc

2006-04-01

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane-similar to the catalytic function of the active site of an enzyme-and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel-scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton-proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein-inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin-K+ channel interactions.

Convergent evolution of chromatin modification by structurally distinct enzymes: comparative enzymology of histone H3 Lys²⁷ methylation by human polycomb repressive complex 2 and vSET.

PubMed

Swalm, Brooke M; Hallenbeck, Kenneth K; Majer, Christina R; Jin, Lei; Scott, Margaret Porter; Moyer, Mikel P; Copeland, Robert A; Wigle, Tim J

2013-07-15

H3K27 (histone H3 Lys27) methylation is an important epigenetic modification that regulates gene transcription. In humans, EZH (enhancer of zeste homologue) 1 and EZH2 are the only enzymes capable of catalysing methylation of H3K27. There is great interest in understanding structure-function relationships for EZH2, as genetic alterations in this enzyme are thought to play a causal role in a number of human cancers. EZH2 is challenging to study because it is only active in the context of the multi-subunit PRC2 (polycomb repressive complex 2). vSET is a viral lysine methyltransferase that represents the smallest protein unit capable of catalysing H3K27 methylation. The crystal structure of this minimal catalytic protein has been solved and researchers have suggested that vSET might prove useful as an EZH2 surrogate for the development of active site-directed inhibitors. To test this proposition, we conducted comparative enzymatic analysis of human EZH2 and vSET and report that, although both enzymes share similar preferences for methylation of H3K27, they diverge in terms of their permissiveness for catalysing methylation of alternative histone lysine sites, their relative preferences for utilization of multimeric macromolecular substrates, their active site primary sequences and, most importantly, their sensitivity to inhibition by drug-like small molecules. The cumulative data led us to suggest that EZH2 and vSET have very distinct active site structures, despite the commonality of the reaction catalysed by the two enzymes. Hence, the EZH2 and vSET pair of enzymes represent an example of convergent evolution in which distinct structural solutions have developed to solve a common catalytic need.

Tree community variation in a tropical continental island according to slope aspect and human interference.

PubMed

Gonçalves, Nathan B; Nettesheim, Felipe C; Conde, Marilena M S

2018-01-01

Associating description of unrecorded tropical tree community structure to sampling approaches that can help determine mechanisms behind floristic variation is important to further the comprehension of how plant species coexist at tropical forests. Thus, this study had the goals of (i) evaluating tree community structure on the continental island of Marambaia (23°4'37.09"S; 43°59'2.15"W) and (ii) testing the prediction that there are local scale changes in a tropical tree community structure between slopes facing different geographic orientation and with distinct human interference history. We established 60 (0.6 ha) sampling units in three different slope sites with distinct predominant geographic orientation and human interference. We sampled all woody trees with diameter at breast height (dbh) ≥ 5 cm. We found a total of 1.170 individuals representing 220 species, 120 genera and 50 families. The overall tree community structure and structural descriptors (abundance of individuals, basal area, species richness and diversity) varied extensively between the sites. The evidence presented here supports that local scale topography variations and human interference history can be important factors contributing to the known floristic heterogeneity of the Atlantic Rainforest. Future work on the study area should focus on disentangling effects from distinct causal factors over tree community variation and species occurrence.

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

PubMed

Deshpande, Ishan; Seeber, Andrew; Shimada, Kenji; Keusch, Jeremy J; Gut, Heinz; Gasser, Susan M

2017-10-19

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Homology modeling, binding site identification and docking study of human angiotensin II type I (Ang II-AT1) receptor.

PubMed

Vyas, Vivek K; Ghate, Manjunath; Patel, Kinjal; Qureshi, Gulamnizami; Shah, Surmil

2015-08-01

Ang II-AT1 receptors play an important role in mediating virtually all of the physiological actions of Ang II. Several drugs (SARTANs) are available, which can block the AT1 receptor effectively and lower the blood pressure in the patients with hypertension. Currently, there is no experimental Ang II-AT1 structure available; therefore, in this study we modeled Ang II-AT1 receptor structure using homology modeling followed by identification and characterization of binding sites and thereby assessing druggability of the receptor. Homology models were constructed using MODELLER and I-TASSER server, refined and validated using PROCHECK in which 96.9% of 318 residues were present in the favoured regions of the Ramachandran plots. Various Ang II-AT1 receptor antagonist drugs are available in the market as antihypertensive drug, so we have performed docking study with the binding site prediction algorithms to predict different binding pockets on the modeled proteins. The identification of 3D structures and binding sites for various known drugs will guide us for the structure-based drug design of novel compounds as Ang II-AT1 receptor antagonists for the treatment of hypertension. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Combining microsatellite, otolith shape and parasites community analyses as a holistic approach to assess population structure of Dentex dentex

NASA Astrophysics Data System (ADS)

Marengo, M.; Baudouin, M.; Viret, A.; Laporte, M.; Berrebi, P.; Vignon, M.; Marchand, B.; Durieux, E. D. H.

2017-10-01

The common dentex, Dentex dentex, is an iconic marine coastal fish of the Mediterranean Sea. Despite its economic and ecological importance, data on the population structure of this species are still very limited. The aim of this study was to identify the stock structure of the common dentex at relatively fine spatial scale around Corsica Island, using a combination of markers that have different spatial and temporal scales of integration: microsatellite DNA markers, otolith shape analysis and parasites communities. Microsatellite analysis indicated that there was no significant genetic differentiation in D. dentex between the four sampling sites around Corsica. Otolith shape analysis suggests one potential distinct population unit of D. dentex centered in one site (Cap Corse) varying in their degree of differentiation from those in the other zones. Multivariate analysis on parasite abundance data highlights to a lower extent two sites (Bonifacio and Galeria) with some connectivity between adjacent zones. The combination of these three markers together highlights the resulting three sites while giving complementary insights and an opportunity to compare their utility and potential to understand population interactions. A complex population structure around Corsican coasts is then proposed, providing a new perspective on common dentex fishery stock conservation and management strategies.

Human prorenin structure sheds light on a novel mechanism of its autoinhibition and on its non-proteolytic activation by the (pro)renin receptor.

PubMed

Morales, Renaud; Watier, Yves; Böcskei, Zsolt

2012-08-03

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor]. Copyright © 2012 Elsevier Ltd. All rights reserved.

Use of magnetic circular dichroism to study dinuclear metallohydrolases and the corresponding biomimetics.

PubMed

Larrabee, James A; Schenk, Gerhard; Mitić, Nataša; Riley, Mark J

2015-09-01

Magnetic circular dichroism (MCD) is a convenient technique for providing structural and mechanistic insight into enzymatic systems in solution. The focus of this review is on aspects of geometric and electronic structure that can be determined by MCD, and how this method can further our understanding of enzymatic mechanisms. Dinuclear Co(II) systems that catalyse hydrolytic reactions were selected to illustrate the approach. These systems all contain active sites with similar structures consisting of two Co(II) ions bridged by one or two carboxylates and a water or hydroxide. In most of these active sites one Co(II) is five-coordinate and one is six-coordinate, with differing binding affinities. It is shown how MCD can be used to determine which binding site--five or six-coordinate--has the greater affinity. Importantly, zero-field-splitting data and magnetic exchange coupling constants may be determined from the temperature and field dependence of MCD data. The relevance of these data to the function of the enzymatic systems is discussed.

High resolution multi-facies realizations of sedimentary reservoir and aquifer analogs

PubMed Central

Bayer, Peter; Comunian, Alessandro; Höyng, Dominik; Mariethoz, Gregoire

2015-01-01

Geological structures are by nature inaccessible to direct observation. This can cause difficulties in applications where a spatially explicit representation of such structures is required, in particular when modelling fluid migration in geological formations. An increasing trend in recent years has been to use analogs to palliate this lack of knowledge, i.e., exploiting the spatial information from sites where the geology is accessible (outcrops, quarry sites) and transferring the observed properties to a study site deemed geologically similar. While this approach is appealing, it is difficult to put in place because of the lack of access to well-documented analog data. In this paper we present comprehensive analog data sets which characterize sedimentary structures from important groundwater hosting formations in Germany and Brazil. Multiple 2-D outcrop faces are described in terms of hydraulic, thermal and chemical properties and interpolated in 3-D using stochastic techniques. These unique data sets can be used by the wider community to implement analog approaches for characterizing reservoir and aquifer formations. PMID:26175910

Structure of the DBL3x domain of pregnancy-associated malaria protein VAR2CSA complexed with chondroitin sulfate A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, K.; Gittis, A.G.; Nguyen, P.

Plasmodium falciparum-infected erythrocytes bind to chondroitin sulfate A (CSA) in the placenta via the VAR2CSA protein, a member of the P. falciparum erythrocyte membrane protein-1 family, leading to life-threatening malaria in pregnant women with severe effects on their fetuses and newborns. Here we describe the structure of the CSA binding DBL3x domain, a Duffy binding-like (DBL) domain of VAR2CSA. By forming a complex of DBL3x with CSA oligosaccharides and determining its structure, we have identified the CSA binding site to be a cluster of conserved positively charged residues on subdomain 2 and subdomain 3. Mutation or chemical modification of lysinemore » residues at the site markedly diminished CSA binding to DBL3x. The location of the CSA binding site is an important step forward in the molecular understanding of pregnancy-associated malaria and offers a new target for vaccine development.« less

Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System*

PubMed Central

Campeotto, Ivan; Percy, Matthew G.; MacDonald, James T.; Förster, Andreas; Freemont, Paul S.; Gründling, Angelika

2014-01-01

Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaPLm and LtaSLm. Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaSLm structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain. PMID:25128528

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

PubMed

Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Comparative analysis of spatial organization of laccases from Cerrena maxima and Coriolus zonatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhukova, Yu. N.; Zhukhlistova, N. E.; Lyashenko, A. V.

2007-09-15

Laccase (oxygen oxidoreductase, EC 1.10.3.2) belongs to the multicopper oxidase family. The main function of this enzyme is to perform electron transfer from the oxidized substrate through the mononuclear copper-containing site T1 to the oxygen molecule bound to the site T3 in the trinuclear T2/T3 cluster. The structures of two new fungal laccases from C. maxima and C. zonatus were solved on the basis of synchrotron X-ray diffraction data. Both laccases show high structural homology with laccases from other sources. The role of the carbohydrate component of laccases in structure stabilization and formation of ordered protein crystals was demonstrated. Inmore » the structures of C. maxima and C. zonatus laccases, two water channels of functional importance were found and characterized. The structural results reported in the present study characterize one of the functional states of the enzyme fixed in the crystal structure.« less

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huerta, Carlos; Borek, Dominika; Machius, Mischa

2009-12-01

Flavin mononucleotide adenylyltransferase (FMNAT) catalyzes the formation of the essential flavocoenzyme flavin adenine dinucleotide (FAD) and plays an important role in flavocoenzyme homeostasis regulation. By sequence comparison, bacterial and eukaryotic FMNAT enzymes belong to two different protein superfamilies and apparently utilize different sets of active-site residues to accomplish the same chemistry. Here we report the first structural characterization of a eukaryotic FMNAT from the pathogenic yeast Candida glabrata. Four crystal structures of C. glabrata FMNAT in different complexed forms were determined at 1.20-1.95 A resolutions, capturing the enzyme active-site states prior to and after catalysis. These structures reveal a novelmore » flavin-binding mode and a unique enzyme-bound FAD conformation. Comparison of the bacterial and eukaryotic FMNATs provides a structural basis for understanding the convergent evolution of the same FMNAT activity from different protein ancestors. Structure-based investigation of the kinetic properties of FMNAT should offer insights into the regulatory mechanisms of FAD homeostasis by FMNAT in eukaryotic organisms.« less

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

PubMed

Ali, Abid; Azam, Mohd W; Khan, Asad U

2018-06-01

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Community structure of aquatic insects in the Esparza River, Costa Rica.

PubMed

Herrera-Vásquez, Jonathan

2009-01-01

This study focused on the structure of the aquatic insect community in spatial and temporal scales in the Esparza River. The river was sampled for one full year throughout 2007. During the dry season low flow months, five sampling points were selected in two different habitats (currents and pools), with five replicates per sample site. During the wet season with peak rain, only the data in the "current habitat" were sampled at each site. Specimens present in the different substrates were collected and preserved in situ. A nested ANOVA was then applied to the data to determine richness and density as the response variables. The variations in temporal and spatial scales were analyzed using width, depth and discharge of the river, and then analyzed using a nested ANOVA. Only a correlation of 51% similarity in richness was found, while in spatial scale, richness showed significant variation between sampling sites, but not between habitats. However, the temporal scale showed significant differences between habitats. Density showed differences between sites and habitats during the dry season in the spatial scale, while in the temporal scale significant variation was found between sampling sites. Width varied between habitats during the dry season, but not between sampling points. Depth showed differences between sampling sites and season. This work studies the importance of community structure of aquatic insects in rivers, and its relevance for the quality of water in rivers and streams.

Macroinvertebrate assemblage recovery following a catastrophic flood and debris flows in an Appalachian mountain stream

USGS Publications Warehouse

Snyder, C.D.; Johnson, Z.B.

2006-01-01

In June 1995, heavy rains caused severe flooding and massive debris flows on the Staunton River, a 3rd-order stream in the Blue Ridge Mountains (Virginia, USA). Scouring caused the loss of the riparian zone and repositioned the stream channel of the lower 2.1 km of the stream. Between 1998 and 2001, we conducted seasonal macroinvertebrate surveys at sites on the Staunton River and on White Oak Canyon Run, a reference stream of similar size and geology that was relatively unaffected by the flood. Our study was designed to determine the extent to which flood-induced changes to the stream channel and riparian habitats caused long-term changes to macroinvertebrate community structure and composition. Sites within the impacted zone of the Staunton River supported diverse stable benthic macroinvertebrate assemblages 3 y after the flood despite dramatic and persistent changes in environmental factors known to be important controls on stream ecosystem function. However, significant differences in total macroinvertebrate density and trophic structure could be attributed to the flood. In autumn, densities of most feeding guilds, including shredders, were higher at impacted-zone sites than at all other sites, suggesting higher overall productivity in the impacted zone. Higher shredder density in the impacted zone was surprising in light of expected decreases in leaf-litter inputs because of removal of riparian forests. In contrast, in spring, we observed density differences in only one feeding guild, scrapers, which showed higher densities at impacted-zone sites than at all other sites. This result conformed to a priori expectations that reduced shading in the impacted zone would lead to increased light and higher instream primary production. We attribute the seasonal differences in trophic structure to the effects of increased temperatures on food quality and to the relationship between the timing of our sampling and the emergence patterns of important taxa. ?? 2006 by The North American Benthological Society.

Investigation of the Binding Profiles of AZD2184 and Thioflavin T with Amyloid-β(1-42) Fibril by Molecular Docking and Molecular Dynamics Methods.

PubMed

Kuang, Guanglin; Murugan, N Arul; Tu, Yaoquan; Nordberg, Agneta; Ågren, Hans

2015-09-03

Detecting deposits of amyloid β fibrils in the brain is of paramount importance for an early diagnosis of Alzheimer's disease. A number of PET tracers have been developed for amyloid imaging, but many suffer from poor specificity and large signal to background ratio. Design of tracers with specificity and improved binding affinity requires knowledge about various potential binding sites in the amyloid β fibril available for the tracers and the nature of the local microenvironment of these sites. In this study we investigate the local structure of fibrils using two important probes, namely, thioflavin T (a fluorescent probe) and AZD2184 (a PET tracer). The target structures for amyloid-β(1-42) fibril are based on reported NMR solution models. By explicitly considering the effect of fibril flexibility on the available binding sites for all these models, the binding affinity of these probes has been investigated. The binding profiles of AZD2184 and thioflavin T were studied by molecular docking and molecular dynamics simulation methods. The two compounds were found to bind at the same sites of the fibril: three of which are within the fibril, and one is on the two sides of the Met35 residue on the surface. The binding affinity of AZD2184 and thioflavin T is found to be higher at the core sites than on the surface due to more contact residues. The binding affinity of AZD2184 is much higher than that of thioflavin T at every site due to electrostatic interaction and spatial restriction, which is in good agreement with experimental observation. However, the structural change of thioflavin T is much more significant than that of AZD2184, which is the chemical basis for its usage as a fluorescent probe. The ramifications of these results for the design and optimization of PET radioligands and fluorescent probes are briefly discussed.

[Effects of land use structure on water quality in Xin'anjiang River].

PubMed

Cao, Fang-Fang; Li, Xue; Wang, Dong; Zhao, Yue; Wang, Yu-Qiu

2013-07-01

Take Xin'anjiang upstream watershed as a case study. Based on data of interpreting TM orthophoto images and water quality monitoring in May 2010, the land use map of Xin'anjiang River, which was categorized to cultivated land, forestland, grassland, water body, building site, was obtained. Using ArcGIS hydrological and spatial analysis function, Xin'anjiang River was divided into eight sub-watersheds, and its watershed land use structure was analyzed. The water quality parameters such as TN, TP, permanganate index, fecal coliform bacteria were monitored from Jan 2010 to Dec 2010. The relations between water quality and land use were analyzed. The results showed that TN and NH4(+) -N had a significant temporal variation: dry season > wet season > normal river flow period, but other parameters did not vary significantly. In the space, Yuliang and Pukou were the most serious pollution sites. Cultivated land, water body, building site had a positive impact on water quality parameters, while there were negative correlation between the forestland and grassland. Annually, cultivated land had the most significantly important effect on TN, NH4(+) -N and permanganate index, and grassland had the most significantly important effect on TP. Cultivated land had the most prominently important impact on water quality parameters in dry season and wet season. What's more, in the normal river flow, cultivated land, grassland and forestland had the most remarkably important influence on TN, TP and fecal coliform bacteria respectively.

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

PubMed

Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

2008-02-22

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gabelli,S.; McLellan, J.; Montalvetti, A.

2006-01-01

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformationalmore » change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.« less

Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malik, Radhika; Viola, Ronald E.

2010-10-28

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identificationmore » of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.« less

Analysis of zinc binding sites in protein crystal structures.

PubMed

Alberts, I L; Nadassy, K; Wodak, S J

1998-08-01

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.

Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

PubMed

Abou-Zied, Osama K

2015-01-01

Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.

Biodiversity in canopy-forming algae: Structure and spatial variability of the Mediterranean Cystoseira assemblages

NASA Astrophysics Data System (ADS)

Piazzi, L.; Bonaviri, C.; Castelli, A.; Ceccherelli, G.; Costa, G.; Curini-Galletti, M.; Langeneck, J.; Manconi, R.; Montefalcone, M.; Pipitone, C.; Rosso, A.; Pinna, S.

2018-07-01

In the Mediterranean Sea, Cystoseira species are the most important canopy-forming algae in shallow rocky bottoms, hosting high biodiverse sessile and mobile communities. A large-scale study has been carried out to investigate the structure of the Cystoseira-dominated assemblages at different spatial scales and to test the hypotheses that alpha and beta diversity of the assemblages, the abundance and the structure of epiphytic macroalgae, epilithic macroalgae, sessile macroinvertebrates and mobile macroinvertebrates associated to Cystoseira beds changed among scales. A hierarchical sampling design in a total of five sites across the Mediterranean Sea (Croatia, Montenegro, Sardinia, Tuscany and Balearic Islands) was used. A total of 597 taxa associated to Cystoseira beds were identified with a mean number per sample ranging between 141.1 ± 6.6 (Tuscany) and 173.9 ± 8.5(Sardinia). A high variability at small (among samples) and large (among sites) scale was generally highlighted, but the studied assemblages showed different patterns of spatial variability. The relative importance of the different scales of spatial variability should be considered to optimize sampling designs and propose monitoring plans of this habitat.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity.

PubMed

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-08-09

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P 3 . Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.

Post-fire tree establishment patterns at the alpine treeline ecotone: Mount Rainier National Park, Washington, USA

Treesearch

Kirk M. Stueve; Dawna L. Cerney; Regina M. Rochefort; Laurie L. Kurth

2009-01-01

Questions: Does tree establishment: (1) occur at a treeline depressed by fire, (2) cause the forest line to ascend upslope, and/or (3) alter landscape heterogeneity? (4) What abiotic and biotic local site conditions are most important in structuring establishment patterns? (5) Does the abiotic setting become more important with increasing upslope distance from the...

Rapid recovery of genetic diversity of dogwhelk (Nucella lapillus L.) populations after local extinction and recolonization contradicts predictions from life-history characteristics.

PubMed

Colson, I; Hughes, R N

2004-08-01

The dogwhelk Nucella lapillus is a predatory marine gastropod populating North Atlantic rocky shores. As with many other gastropod species, N. lapillus was affected by tributyltin (TBT) pollution during the 1970s and 1980s, when local populations became extinct. After a partial ban on TBT in the United Kingdom in 1987, vacant sites have been recolonized. N. lapillus lacks a planktonic larval stage and is therefore expected to have limited dispersal ability. Relatively fast recolonization of some sites, however, contradicts this assumption. We compared levels of genetic diversity and genetic structuring between recolonized sites and sites that showed continuous population at three localities across the British Isles. No significant genetic effects of extinction/recolonization events were observed in SW Scotland and NE England. In SW England we observed a decrease in genetic diversity and an increase in genetic structure in recolonized populations. This last result could be an artefact, however, due to the superposition of other local factors influencing the genetic structuring of dogwhelk populations. We conclude that recolonization of vacant sites was accomplished by a relatively high number of individuals originating from several source populations (the 'migrant-pool' model of recolonization), implying that movements are more widespread than expected on the basis of development mode alone. Comparison with published data on genetic structure of marine organisms with contrasted larval dispersal supports this hypothesis. Our results also stress the importance of local factors (geographical or ecological) in determining genetic structure of dogwhelk populations. Copyright 2004 Blackwell Publishing Ltd

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

PubMed

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

Janssen, Bert J C; Christodoulidou, Agni; McCarthy, Andrew; Lambris, John D; Gros, Piet

2006-11-09

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

PubMed Central

Garvin, Michael R.; Bielawski, Joseph P.; Gharrett, Anthony J.

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm. PMID:21969854

Positive Darwinian selection in the piston that powers proton pumps in complex I of the mitochondria of Pacific salmon.

PubMed

Garvin, Michael R; Bielawski, Joseph P; Gharrett, Anthony J

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm.

The role of conserved surface hydrophobic residues in the carbapenemase activity of the class D β-lactamases.

PubMed

Toth, Marta; Smith, Clyde A; Antunes, Nuno T; Stewart, Nichole K; Maltz, Lauren; Vakulenko, Sergei B

2017-08-01

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) produce resistance to the last-resort carbapenem antibiotics and render these drugs ineffective for the treatment of life-threatening infections. Here, it is shown that among the clinically important CHDLs, OXA-143 produces the highest levels of resistance to carbapenems and has the highest catalytic efficiency against these substrates. Structural data demonstrate that acylated carbapenems entirely fill the active site of CHDLs, leaving no space for water molecules, including the deacylating water. Since the entrance to the active site is obstructed by the acylated antibiotic, the deacylating water molecule must take a different route for entry. It is shown that in OXA-143 the movement of a conserved hydrophobic valine residue on the surface opens a channel to the active site of the enzyme, which would not only allow the exchange of water molecules between the active site and the milieu, but would also create extra space for a water molecule to position itself in the vicinity of the scissile bond of the acyl-enzyme intermediate to perform deacylation. Structural analysis of the OXA-23 carbapenemase shows that in this enzyme movement of the conserved leucine residue, juxtaposed to the valine on the molecular surface, creates a similar channel to the active site. These data strongly suggest that all CHDLs may employ a mechanism whereupon the movement of highly conserved valine or leucine residues would allow a water molecule to access the active site to promote deacylation. It is further demonstrated that the 6α-hydroxyethyl group of the bound carbapenem plays an important role in the stabilization of this channel. The recognition of a universal deacylation mechanism for CHDLs suggests a direction for the future development of inhibitors and novel antibiotics for these enzymes of utmost clinical importance.

Structural signatures of the complex formed between 3-nitro-4-hydroxybenzoate and the Zn(II)-substituted R6 insulin hexamer

PubMed Central

Olsen, Helle Birk; Leuenberger-Fisher, Melissa R.; Kadima, Webe; Borchardt, Dan; Kaarsholm, Niels C.; Dunn, Michael F.

2003-01-01

3-Nitro-4-hydroxybenzoate (3N4H) is a probe of the structure and dynamics of the metal-centered His B10 assembly sites of the insulin hexamer. Each His B10 site consists of a ∼12 Å-long cavity situated on the threefold symmetry axis. These sites play an important role in the storage and release of insulin in vivo. The allosteric behavior of the insulin hexamer is modulated by ligand binding to the His B10 zinc sites and to the phenolic pockets. Binding to these sites drives transitions among three allosteric states, designated T6, T3R3, and R6. Although a wide variety of mono anions bind to the His B10 zinc sites of R3, X-ray structures of ligands complexed to this site exist only for H2O, Cl–, and SCN–. This work combines one- and two-dimensional 1H NMR and UV-Vis absorbance studies of the structure and dynamics of the 3N4H complex, which establish the following: (1) relative to the NMR time scale, 3N4H exchange between free and bound states is slow, while flipping among three equivalent orientations about the site threefold axis is fast; (2) binding of 3N4H perturbs resonances within the His B10 zinc site and generates NOEs between ligand resonances and the insulin C-α and side chain resonances of ValB2, AsnB3, LeuB6, and CysB7; and (3) 3N4H exchange for other ligands is limited by a protein conformational transition. These results are consistent with coordination of the 3N4H carboxylate to the His B10 zinc ion and van der Waals interactions with Val B2, Asn B3, Leu B6, and Cys A7. PMID:12930990

A simple and efficient method for predicting protein-protein interaction sites.

PubMed

Higa, R H; Tozzi, C L

2008-09-23

Computational methods for predicting protein-protein interaction sites based on structural data are characterized by an accuracy between 70 and 80%. Some experimental studies indicate that only a fraction of the residues, forming clusters in the center of the interaction site, are energetically important for binding. In addition, the analysis of amino acid composition has shown that residues located in the center of the interaction site can be better discriminated from the residues in other parts of the protein surface. In the present study, we implement a simple method to predict interaction site residues exploiting this fact and show that it achieves a very competitive performance compared to other methods using the same dataset and criteria for performance evaluation (success rate of 82.1%).

Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patterson, Edward I.; EH Graham Centre for Agricultural Innovation; Dombrovski, Andrew K.

2013-09-06

Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediatemore » nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface.« less

A general strategy to solve the phase problem in RNA crystallography

PubMed Central

Keel, Amanda Y.; Rambo, Robert P.; Batey, Robert T.; Kieft, Jeffrey S.

2007-01-01

SUMMARY X-ray crystallography of biologically important RNA molecules has been hampered by technical challenges, including finding a heavy-atom derivative to obtain high-quality experimental phase information. Existing techniques have drawbacks, severely limiting the rate at which important new structures are solved. To address this need, we have developed a reliable means to localize heavy atoms specifically to virtually any RNA. By solving the crystal structures of thirteen variants of the G·U wobble pair cation binding motif we have identified an optimal version that when inserted into an RNA helix introduces a high-occupancy cation binding site suitable for phasing. This “directed soaking” strategy can be integrated fully into existing RNA and crystallography methods, potentially increasing the rate at which important structures are solved and facilitating routine solving of structures using Cu-Kα radiation. The success of this method has been proven in that it has already been used to solve several novel crystal structures. PMID:17637337

Post-fire regeneration in seasonally dry tropical forest fragments in southeastern Brazil.

PubMed

Costa, Mayke B; Menezes, Luis Fernando T DE; Nascimento, Marcelo T

2017-01-01

Seasonally dry tropical forest is one of the highly threatened biome. However, studies on the effect of fire on these tree communities are still scarce. In this context, a floristic and structural survey in three forest areas in the southeast of Brazil that were affected by fire between 14 and 25 years ago was performed with the objective of evaluating post-fire regeneration. In each site, five systematically placed plots (25 m x 25 m each) were established. The more recently burnt site had significantly lower values of richness and diversity than the other two sites. However, the sites did not differ in density and basal area. Annona dolabripetala, Astronium concinnum, Joannesia princeps and Polyandrococos caudescens were within the 10 most important species for the three sites. Comparing these data with adjacent mature forests, the results indicated differences both in structural and floristic aspects, suggesting that the time after fire was not sufficient for recuperation of these areas. The recovery process indicate at least 190 years for areas return to basal area values close to those observed in mature forests nearby.

Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

PubMed Central

Codoñer, Francisco M.; Fares, Mario A.; Elena, Santiago F.

2006-01-01

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions. PMID:16731922

Adaptive covariation between the coat and movement proteins of prunus necrotic ringspot virus.

PubMed

Codoñer, Francisco M; Fares, Mario A; Elena, Santiago F

2006-06-01

The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.

Dihydrofolate reductase: A potential drug target in trypanosomes and leishmania

NASA Astrophysics Data System (ADS)

Zuccotto, Fabio; Martin, Andrew C. R.; Laskowski, Roman A.; Thornton, Janet M.; Gilbert, Ian H.

1998-05-01

Dihydrofolate reductase has successfully been used as a drug target in the area of anti-cancer, anti-bacterial and anti-malarial chemotherapy. Little has been done to evaluate it as a drug target for treatment of the trypanosomiases and leishmaniasis. A crystal structure of Leishmania major dihydrofolate reductase has been published. In this paper, we describe the modelling of Trypanosoma cruzi and Trypanosoma brucei dihydrofolate reductases based on this crystal structure. These structures and models have been used in the comparison of protozoan, bacterial and human enzymes in order to highlight the different features that can be used in the design of selective anti-protozoan agents. Comparison has been made between residues present in the active site, the accessibility of these residues, charge distribution in the active site, and the shape and size of the active sites. Whilst there is a high degree of similarity between protozoan, human and bacterial dihydrofolate reductase active sites, there are differences that provide potential for selective drug design. In particular, we have identified a set of residues which may be important for selective drug design and identified a larger binding pocket in the protozoan than the human and bacterial enzymes.

Computational characterization of chromatin domain boundary-associated genomic elements

PubMed Central

Hong, Seungpyo

2017-01-01

Abstract Topologically associated domains (TADs) are 3D genomic structures with high internal interactions that play important roles in genome compaction and gene regulation. Their genomic locations and their association with CCCTC-binding factor (CTCF)-binding sites and transcription start sites (TSSs) were recently reported. However, the relationship between TADs and other genomic elements has not been systematically evaluated. This was addressed in the present study, with a focus on the enrichment of these genomic elements and their ability to predict the TAD boundary region. We found that consensus CTCF-binding sites were strongly associated with TAD boundaries as well as with the transcription factors (TFs) Zinc finger protein (ZNF)143 and Yin Yang (YY)1. TAD boundary-associated genomic elements include DNase I-hypersensitive sites, H3K36 trimethylation, TSSs, RNA polymerase II, and TFs such as Specificity protein 1, ZNF274 and SIX homeobox 5. Computational modeling with these genomic elements suggests that they have distinct roles in TAD boundary formation. We propose a structural model of TAD boundaries based on these findings that provides a basis for studying the mechanism of chromatin structure formation and gene regulation. PMID:28977568

Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong

2009-11-10

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenzamore » H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.« less

Fortified Settlements of the 9th and 10th Centuries ad in Central Europe: Structure, Function and Symbolism

PubMed Central

Herold, Hajnalka

2012-01-01

THE STRUCTURE, FUNCTION(S) and symbolism of early medieval (9th–10th centuries ad) fortified settlements from central Europe, in particular today’s Austria, Hungary, Czech Republic and Slovakia, are examined in this paper. It offers an overview of the current state of research together with new insights based on analysis of the site of Gars-Thunau in Lower Austria. Special emphasis is given to the position of the fortified sites in the landscape, to the elements of the built environment and their spatial organisation, as well as to graves within the fortified area. The region under study was situated on the SE border of the Carolingian (and later the Ottonian) Empire, with some of the discussed sites lying in the territory of the ‘Great Moravian Empire’ in the 9th and 10th centuries. These sites can therefore provide important comparative data for researchers working in other parts of the Carolingian Empire and neighbouring regions. PMID:23564981

Cloud Computing for Protein-Ligand Binding Site Comparison

PubMed Central

2013-01-01

The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery. PMID:23762824

Cloud computing for protein-ligand binding site comparison.

PubMed

Hung, Che-Lun; Hua, Guan-Jie

2013-01-01

The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

The crystal structure of a cardiovirus RNA-dependent RNA polymerase reveals an unusual conformation of the polymerase active site.

PubMed

Vives-Adrian, Laia; Lujan, Celia; Oliva, Baldo; van der Linden, Lonneke; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; van Kuppeveld, Frank J M; Ferrer-Orta, Cristina; Verdaguer, Núria

2014-05-01

Encephalomyocarditis virus (EMCV) is a member of the Cardiovirus genus within the large Picornaviridae family, which includes a number of important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for viral genome replication. In this study, we report the X-ray structures of two different crystal forms of the EMCV RdRp determined at 2.8- and 2.15-Å resolution. The in vitro elongation and VPg uridylylation activities of the purified enzyme have also been demonstrated. Although the overall structure of EMCV 3Dpol is shown to be similar to that of the known RdRps of other members of the Picornaviridae family, structural comparisons show a large reorganization of the active-site cavity in one of the crystal forms. The rearrangement affects mainly motif A, where the conserved residue Asp240, involved in ribonucleoside triphosphate (rNTP) selection, and its neighbor residue, Phe239, move about 10 Å from their expected positions within the ribose binding pocket toward the entrance of the rNTP tunnel. This altered conformation of motif A is stabilized by a cation-π interaction established between the aromatic ring of Phe239 and the side chain of Lys56 within the finger domain. Other contacts, involving Phe239 and different residues of motif F, are also observed. The movement of motif A is connected with important conformational changes in the finger region flanked by residues 54 to 63, harboring Lys56, and in the polymerase N terminus. The structures determined in this work provide essential information for studies on the cardiovirus RNA replication process and may have important implications for the development of new antivirals targeting the altered conformation of motif A. The Picornaviridae family is one of the largest virus families known, including many important human and animal pathogens. The RNA-dependent RNA polymerase (RdRp) 3Dpol is a key enzyme for picornavirus genome replication and a validated target for the development of antiviral therapies. Solving the X-ray structure of the first cardiovirus RdRp, EMCV 3Dpol, we captured an altered conformation of a conserved motif in the polymerase active site (motif A) containing the aspartic acid residue involved in rNTP selection and binding. This altered conformation of motif A, which interferes with the correct positioning of the rNTP substrate in the active site, is stabilized by a number of residues strictly conserved among picornaviruses. The rearrangements observed suggest that this motif A segment is a dynamic element that can be modulated by external effectors, either activating or inhibiting enzyme activity, and this type of modulation appears to be general to all picornaviruses.

10 CFR 72.122 - Overall requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... natural phenomena. (1) Structures, systems, and components important to safety must be designed to... effects of natural phenomena such as earthquakes, tornadoes, lightning, hurricanes, floods, tsunami, and... severe of the natural phenomena reported for the site and surrounding area, with appropriate margins to...

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

[Computational chemistry in structure-based drug design].

PubMed

Cao, Ran; Li, Wei; Sun, Han-Zi; Zhou, Yu; Huang, Niu

2013-07-01

Today, the understanding of the sequence and structure of biologically relevant targets is growing rapidly and researchers from many disciplines, physics and computational science in particular, are making significant contributions to modern biology and drug discovery. However, it remains challenging to rationally design small molecular ligands with desired biological characteristics based on the structural information of the drug targets, which demands more accurate calculation of ligand binding free-energy. With the rapid advances in computer power and extensive efforts in algorithm development, physics-based computational chemistry approaches have played more important roles in structure-based drug design. Here we reviewed the newly developed computational chemistry methods in structure-based drug design as well as the elegant applications, including binding-site druggability assessment, large scale virtual screening of chemical database, and lead compound optimization. Importantly, here we address the current bottlenecks and propose practical solutions.

In silico investigation into the interactions between murine 5-HT3 receptor and the principle active compounds of ginger (Zingiber officinale).

PubMed

Lohning, Anna E; Marx, Wolfgang; Isenring, Liz

2016-11-01

Gingerols and shogaols are the primary non-volatile actives within ginger (Zingiber officinale). These compounds have demonstrated in vitro to exert 5-HT 3 receptor antagonism which could benefit chemotherapy-induced nausea and vomiting (CINV). The site and mechanism of action by which these compounds interact with the 5-HT 3 receptor is not fully understood although research indicates they may bind to a currently unidentified allosteric binding site. Using in silico techniques, such as molecular docking and GRID analysis, we have characterized the recently available murine 5-HT 3 receptor by identifying sites of strong interaction with particular functional groups at both the orthogonal (serotonin) site and a proposed allosteric binding site situated at the interface between the transmembrane region and the extracellular domain. These were assessed concurrently with the top-scoring poses of the docked ligands and included key active gingerols, shogaols and dehydroshogaols as well as competitive antagonists (e.g. setron class of pharmacologically active drugs), serotonin and its structural analogues, curcumin and capsaicin, non-competitive antagonists and decoys. Unexpectedly, we found that the ginger compounds and their structural analogs generally outscored other ligands at both sites. Our results correlated well with previous site-directed mutagenesis studies in identifying key binding site residues. We have identified new residues important for binding the ginger compounds. Overall, the results suggest that the ginger compounds and their structural analogues possess a high binding affinity to both sites. Notwithstanding the limitations of such theoretical analyses, these results suggest that the ginger compounds could act both competitively or non-competitively as has been shown for palonosetron and other modulators of CYS loop receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Indel PDB: a database of structural insertions and deletions derived from sequence alignments of closely related proteins.

PubMed

Hsing, Michael; Cherkasov, Artem

2008-06-25

Insertions and deletions (indels) represent a common type of sequence variations, which are less studied and pose many important biological questions. Recent research has shown that the presence of sizable indels in protein sequences may be indicative of protein essentiality and their role in protein interaction networks. Examples of utilization of indels for structure-based drug design have also been recently demonstrated. Nonetheless many structural and functional characteristics of indels remain less researched or unknown. We have created a web-based resource, Indel PDB, representing a structural database of insertions/deletions identified from the sequence alignments of highly similar proteins found in the Protein Data Bank (PDB). Indel PDB utilized large amounts of available structural information to characterize 1-, 2- and 3-dimensional features of indel sites. Indel PDB contains 117,266 non-redundant indel sites extracted from 11,294 indel-containing proteins. Unlike loop databases, Indel PDB features more indel sequences with secondary structures including alpha-helices and beta-sheets in addition to loops. The insertion fragments have been characterized by their sequences, lengths, locations, secondary structure composition, solvent accessibility, protein domain association and three dimensional structures. By utilizing the data available in Indel PDB, we have studied and presented here several sequence and structural features of indels. We anticipate that Indel PDB will not only enable future functional studies of indels, but will also assist protein modeling efforts and identification of indel-directed drug binding sites.

Effect of Land Cover Type and Structure on Water Cycling Dynamics for Agricultural and Wetland Sites in the Sacramento/San Joaquin River Delta

NASA Astrophysics Data System (ADS)

Eichelmann, E.; Hemes, K. S.; Baldocchi, D. D.

2016-12-01

The Sacramento/San Joaquin river delta is an important source of fresh water for California. To reverse soil subsidence, which is linked to draining the natural wetlands for agriculture, parts of the Sacramento/San Joaquin river delta have been restored to managed wetlands. While these restored wetlands provide greenhouse gas benefits compared to agricultural use of the land, implications for the water balance of these ecosystems, specifically evapotranspiration, are not well known. Based on multiple years of eddy covariance measurements of water, CO2, and sensible energy fluxes we explored the water cycling dynamics for several sites under different land use covers in the Sacramento/San Joaquin river delta. We investigated four sites under agricultural use (rice, corn, and alfalfa crops and cow pasture) and three restored wetland sites of varying ages and structures to examine the influence of land cover type and structure on evapotranspiration, sensible energy flux, and water use efficiency. While the wetland and the rice sites are usually flooded for the majority of the year, the alfalfa, corn, and pasture sites have a water table that is maintained to be below ground level throughout the year. The three wetland sites also have different fractions of open water to vegetation, covering a gradient from very dense vegetation with no open water to a fairly open structure with large pools of open water. These differences in land cover (dry vs flooded and fraction of open water to vegetation) have an effect on the patterns of evapotranspiration on diurnal to annual timescales. Although the flooded sites (wetland sites and rice) tend to have larger annual evapotranspiration than the drained sites (cow pasture, alfalfa, and corn), the fraction of open water to vegetation affects the extend to which the flooded sites' evapotranspiration exceeds that of drained sites. On diurnal timescales, we found that flooded sites with a larger fraction of open water to vegetation have larger night time latent energy fluxes, especially during the first half of the night. In contrast, flooded sites with a lower open water fraction have lower night time latent energy fluxes which are comparable in magnitude to night time fluxes at drained sites.

Superpredator proximity and landscape characteristics alters nest site selection and breeding success of a subordinate predator.

PubMed

Atuo, Fidelis Akunke; O'Connell, Timothy John

2018-03-01

Selecting nesting habitat that minimizes predation risk but maximizes foraging success is one of the most important decisions in avian life history. This takes on added complexity when a predator is faced with the challenge of avoiding fellow predators. We assessed the importance of local and landscape vegetation, food abundance, and predation risk on nest site selection and nest survival in a subordinate raptor (Mississippi Kite; Ictinia mississippiensis) nesting in proximity to two superpredators, Red-tailed hawk (Buteo jamaicensis) and Great horned owl (Bubo virginianus). All three species nested in trees in a grassland landscape. In this landscape, kites favored upland trees and shrubs, avoiding their more typical riparian forest association elsewhere in the species' range. Compared to random conditions, kites selected nest sites with high tree density and more closed canopy in the surrounding area. Mississippi Kite selection was not related to food abundance but could be explained by the presence of superpredators (i.e., hawks and owls) selecting riparian woodland for their nests. Nest survival declined with proximity to superpredator nesting sites. Overall, our study demonstrates how landscape structure and superior predators shapes predation risk for subordinate predators. Our results emphasize the importance of spatial heterogeneity in presenting opportunities for subordinate predators to coexist in a landscape with important superpredators.

Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

PubMed Central

Rajasekaran, M.; Abirami, Santhanam; Chen, Chinpan

2011-01-01

Background Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. Methodology/Principal Findings We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. Conclusions/Significance Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms. PMID:21980537

Geomfinder: a multi-feature identifier of similar three-dimensional protein patterns: a ligand-independent approach.

PubMed

Núñez-Vivanco, Gabriel; Valdés-Jiménez, Alejandro; Besoaín, Felipe; Reyes-Parada, Miguel

2016-01-01

Since the structure of proteins is more conserved than the sequence, the identification of conserved three-dimensional (3D) patterns among a set of proteins, can be important for protein function prediction, protein clustering, drug discovery and the establishment of evolutionary relationships. Thus, several computational applications to identify, describe and compare 3D patterns (or motifs) have been developed. Often, these tools consider a 3D pattern as that described by the residues surrounding co-crystallized/docked ligands available from X-ray crystal structures or homology models. Nevertheless, many of the protein structures stored in public databases do not provide information about the location and characteristics of ligand binding sites and/or other important 3D patterns such as allosteric sites, enzyme-cofactor interaction motifs, etc. This makes necessary the development of new ligand-independent methods to search and compare 3D patterns in all available protein structures. Here we introduce Geomfinder, an intuitive, flexible, alignment-free and ligand-independent web server for detailed estimation of similarities between all pairs of 3D patterns detected in any two given protein structures. We used around 1100 protein structures to form pairs of proteins which were assessed with Geomfinder. In these analyses each protein was considered in only one pair (e.g. in a subset of 100 different proteins, 50 pairs of proteins can be defined). Thus: (a) Geomfinder detected identical pairs of 3D patterns in a series of monoamine oxidase-B structures, which corresponded to the effectively similar ligand binding sites at these proteins; (b) we identified structural similarities among pairs of protein structures which are targets of compounds such as acarbose, benzamidine, adenosine triphosphate and pyridoxal phosphate; these similar 3D patterns are not detected using sequence-based methods; (c) the detailed evaluation of three specific cases showed the versatility of Geomfinder, which was able to discriminate between similar and different 3D patterns related to binding sites of common substrates in a range of diverse proteins. Geomfinder allows detecting similar 3D patterns between any two pair of protein structures, regardless of the divergency among their amino acids sequences. Although the software is not intended for simultaneous multiple comparisons in a large number of proteins, it can be particularly useful in cases such as the structure-based design of multitarget drugs, where a detailed analysis of 3D patterns similarities between a few selected protein targets is essential.

[Peculiarities of secondary structure of serum albumin of some representatives of the animal kingdom].

PubMed

Pekhymenko, G V; Kuchmerovskaia, T M

2011-01-01

Methods of infrared (IR) spectroscopy and circular dichroism (CD) are suitable techniques for detection of proteins structural changes. These methods were used for determinating peculiarities of the secondary structure of serum albumins in some representatives of two classes of reptiles: Horsfield's tortoise (Testudo horsfieldi), water snake (Natrix tessellata) and grass snake (Natrix natrix) and birds: domestic goose (Anser anser), domestic chicken (Gallus domesticus), domestic duck (Anas platyrhyncha) and dove colored (Columba livia). An analysis of IR spectra and spectra obtained by the method of CD of serum albumins of both classes representatives revealed that beta-folding structure and alpha-helical sections that form the alpha-conformation play an important role in conformational structure formation of polypeptide chain and also disordered sites of molecules of these proteins. It was observed that certain redistribution depending on animals species exists, in the formation of secondary structure of serum albumins of the investigated representatives of reptiles and birds classes between the content of beta-folding structure, alpha-helical sections and disordered sites in molecules of these proteins.

Polyamide membranes with nanoscale Turing structures for water purification

NASA Astrophysics Data System (ADS)

Tan, Zhe; Chen, Shengfu; Peng, Xinsheng; Zhang, Lin; Gao, Congjie

2018-05-01

The emergence of Turing structures is of fundamental importance, and designing these structures and developing their applications have practical effects in chemistry and biology. We use a facile route based on interfacial polymerization to generate Turing-type polyamide membranes for water purification. Manipulation of shapes by control of reaction conditions enabled the creation of membranes with bubble or tube structures. These membranes exhibit excellent water-salt separation performance that surpasses the upper-bound line of traditional desalination membranes. Furthermore, we show the existence of high water permeability sites in the Turing structures, where water transport through the membranes is enhanced.

CASTp 3.0: computed atlas of surface topography of proteins.

PubMed

Tian, Wei; Chen, Chang; Lei, Xue; Zhao, Jieling; Liang, Jie

2018-06-01

Geometric and topological properties of protein structures, including surface pockets, interior cavities and cross channels, are of fundamental importance for proteins to carry out their functions. Computed Atlas of Surface Topography of proteins (CASTp) is a web server that provides online services for locating, delineating and measuring these geometric and topological properties of protein structures. It has been widely used since its inception in 2003. In this article, we present the latest version of the web server, CASTp 3.0. CASTp 3.0 continues to provide reliable and comprehensive identifications and quantifications of protein topography. In addition, it now provides: (i) imprints of the negative volumes of pockets, cavities and channels, (ii) topographic features of biological assemblies in the Protein Data Bank, (iii) improved visualization of protein structures and pockets, and (iv) more intuitive structural and annotated information, including information of secondary structure, functional sites, variant sites and other annotations of protein residues. The CASTp 3.0 web server is freely accessible at http://sts.bioe.uic.edu/castp/.

Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

NASA Astrophysics Data System (ADS)

Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

2015-12-01

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

Variability of site response in the Los Angeles urban area

USGS Publications Warehouse

Hartzell, S.; Cranswick, E.; Frankel, A.; Carver, D.; Meremonte, M.

1997-01-01

This article addresses the variability of site response in the Los Angeles area and possible structural causes for the observations. Aftershock records from 231 sites in the San Fernando and Los Angeles basins and the surrounding mountains are used in this study. Spectral ratios, taken with respect to a low-amplitude reference site, are used to document the variation in site amplification in the frequency range 2 to 6 Hz, both spatially and with backazimuth to the source. At higher frequencies (6 to 10 Hz), spectral ratios are shown to have greater spatial variability. Interstation spectral ratios are used to measure the standard deviation among sources as a function of station separation. An increase in the variation in ground motion is shown to take place at a station separation of 1 km. Relative site-response estimates between nearby stations are used to demonstrate that preferred directions of motion can exist even in areas with no surface topographic effects. Significant variations in site response exist over short baselines (up to a factor of 2 over 200 m) that are not explained by differences in surficial geology or shallow shear-wave velocity. A variety of investigative approaches is used, including spectral ratios, arrival-time variations, 1D and 2D waveform modeling, and comparison with seismic reflection lines, to determine the most likely causes of these observations. A correlation is demonstrated between late arrival times of P and S waves and larger site amplification in Sherman Oaks and Northridge. This observation, in conjunction with waveform modeling and seismic reflection profiles, is used to infer that sedimentary structures in the upper 1 to 2 km and topography on the sediment-basement interface play an important role in determining site amplification. These structures, in the form of folds and buried basins, focus energy in spatially restricted areas at the surface. Comparison of displacement waveforms at nearby stations having disparate site amplifications, complemented by known shallow shear-wave velocities at selected sites, is used to support the argument that these structures, in some cases, can be the dominant factor in the modification of local ground motions.

A Study to Understand the Potential Vulnerabilities to the Foundations of Historic Structures in Coastal Areas

NASA Astrophysics Data System (ADS)

Hall, C. L.; Curran, B.; Jefferson, M.; Routhier, M.; Mulukutla, G. K.

2011-12-01

Sea level rise, one of the most important manifestations of climate change is expected to result in increased coastal erosion and storm surge flooding. This work reports on a project undertaken to assess the vulnerability of foundations of historic structures in coastal areas to the potential consequences from climate change resulting from increased storm surge flooding. Foundations of historic buildings are especially vulnerable to the seepage and ground water intrusion. Many historic structures themselves are used to house valuable historic collections making it all the important to preserve and protect these structures. Data collected from a field subsurface geophysical survey, geospatial field survey, and a simulation to understand the hydrological conditions of the site useful to build an understanding of threats to foundations of historic structures located on the coast. The study site chosen for this project is the Strawbery Banke Living History Museum, in Portsmouth, NH. Located very close to the banks of the Piscataqua River at its meeting point with the Atlantic Ocean, it consists of many buildings of historical importance dating as far back as 1690. Upstream of the river is the tidal estuary system, the Great Bay which itself is formed by a confluence of seven major rivers, making it a highly dynamic, tide-dominated body of fresh and saltwater. Strawbery Banke is representative of historic structures, located on the Northeastern coast of the US, that will likely be impacted by climate change. The primary threat to the sub-structures for the site is Puddle Dock; a tidal inlet , that lies at the heart of the facility, that once provided direct river access to Strawbery Banke (and since filled around 1900). Results from the long term deployment of in -situ water level loggers in test wells, temperature and relative humidity sensors, and a ground penetration radar survey of the Puddle Dock, along with the detailed fine-scale elevation survey of the site provide a window into the seasonality of ground water levels and the dynamic equilibrium of fresh and saltwater. Initial analysis of the data indicates that at present Puddle Dock, acts as a conduit for tidal flows, albeit in a restrained manner. This can be attributed to the very unscientific way that it was filled perhaps with garbage, and other waste. It is anticipated the principles applied for this work can be replicated for assessing the vulnerability of other at-risk historical structures in the Northeastern US. Support for purchase of hardware came from National Geographic Society's Waitt Grant program, and the Undergraduate scholarships support was provided by a grant from University of New Hampshire's Joan and James Leitzel Center for Mathematics, Science and Engineering Education.

Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1).

PubMed

Asciutto, Eliana K; Pochapsky, Thomas C

2018-04-27

Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H- 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Technological advances in site-directed spin labeling of proteins.

PubMed

Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

2013-10-01

Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Crystal structure of the Tum1 protein from the yeast Saccharomyces cerevisiae.

PubMed

Qiu, Rui; Wang, Fengbin; Liu, Meiruo; Lou, Tiantian; Ji, Chaoneng

2012-11-01

Yeast tRNA-thiouridine modification protein 1 (Tum1) plays essential role in the sulfur transfer process of Urm1 system, which in turn is involved in many important cellular processes. In the rhodanese-like domain (RLD), conserved cysteine residue is proved to be the centre of active site of sulfurtransferases and crucial for the substrate recognition. In this report, we describe the crystal structure of Tum1 protein at 1.90 A resolution which, despite consisting of two RLDs, has only one conserved cysteine residue in the C-terminal RLD. An unaccounted electron density is found near the active site, which might point to the new cofactor in the sulfur transfer mechanism.

Solid-state NMR studies of form I of atorvastatin calcium.

PubMed

Wang, Wei David; Gao, Xudong; Strohmeier, Mark; Wang, Wei; Bai, Shi; Dybowski, Cecil

2012-03-22

Solid-state (13)C, (19)F, and (15)N magic angle spinning NMR studies of Form I of atorvastatin calcium are reported, including chemical shift tensors of all resolvable carbon sites and fluorine sites. The complete (13)C and (19)F chemical shift assignments are given based on an extensive analysis of (13)C-(1)H HETCOR and (13)C-(19)F HETCOR results. The solid-state NMR data indicate that the asymmetric unit of this material contains two atorvastatin molecules. A possible structure of Form I of atorvastatin calcium (ATC-I), derived from solid-state NMR data and density functional theory calculations of various structures, is proposed for this important active pharmaceutical ingredient (API).

Identifying selectively important amino acid positions associated with alternative habitat environments in fish mitochondrial genomes.

PubMed

Xia, Jun Hong; Li, Hong Lian; Zhang, Yong; Meng, Zi Ning; Lin, Hao Ran

2018-05-01

Fish species inhabitating seawater (SW) or freshwater (FW) habitats have to develop genetic adaptations to alternative environment factors, especially salinity. Functional consequences of the protein variations associated with habitat environments in fish mitochondrial genomes have not yet received much attention. We analyzed 829 complete fish mitochondrial genomes and compared the amino acid differences of 13 mitochondrial protein families between FW and SW fish groups. We identified 47 specificity determining sites (SDS) that associated with FW or SW environments from 12 mitochondrial protein families. Thirty-two (68%) of the SDS sites are hydrophobic, 13 (28%) are neutral, and the remaining sites are acidic or basic. Seven of those SDS from ND1, ND2 and ND5 were scored as probably damaging to the protein structures. Furthermore, phylogenetic tree based Bayes Empirical Bayes analysis also detected 63 positive sites associated with alternative habitat environments across ten mtDNA proteins. These signatures could be important for studying mitochondrial genetic variation relevant to fish physiology and ecology.

Plant Communities Rather than Soil Properties Structure Arbuscular Mycorrhizal Fungal Communities along Primary Succession on a Mine Spoil

PubMed Central

Krüger, Claudia; Kohout, Petr; Janoušková, Martina; Püschel, David; Frouz, Jan; Rydlová, Jana

2017-01-01

Arbuscular mycorrhizal fungal (AMF) community assembly during primary succession has so far received little attention. It remains therefore unclear, which of the factors, driving AMF community composition, are important during ecosystem development. We addressed this question on a large spoil heap, which provides a mosaic of sites in different successional stages under different managements. We selected 24 sites of c. 12, 20, 30, or 50 years in age, including sites with spontaneously developing vegetation and sites reclaimed by alder plantations. On each site, we sampled twice a year roots of the perennial rhizomatous grass Calamagrostis epigejos (Poaceae) to determine AMF root colonization and diversity (using 454-sequencing), determined the soil chemical properties and composition of plant communities. AMF taxa richness was unaffected by site age, but AMF composition variation increased along the chronosequences. AMF communities were unaffected by soil chemistry, but related to the composition of neighboring plant communities of the sampled C. epigejos plants. In contrast, the plant communities of the sites were more distinctively structured than the AMF communities along the four successional stages. We conclude that AMF and plant community successions respond to different factors. AMF communities seem to be influenced by biotic rather than by abiotic factors and to diverge with successional age. PMID:28473828

How to Create a Navigational Wireframe in Word, With Site Map Example

EPA Pesticide Factsheets

Use Microsoft Word's graphic tools to create a wireframe: an organization chart showing the top three levels of HTML content (Home page, secondary pages, and tertiary pages). This is an important step in planning the structure of a website.

Computational Tools for Allosteric Drug Discovery: Site Identification and Focus Library Design.

PubMed

Huang, Wenkang; Nussinov, Ruth; Zhang, Jian

2017-01-01

Allostery is an intrinsic phenomenon of biological macromolecules involving regulation and/or signal transduction induced by a ligand binding to an allosteric site distinct from a molecule's active site. Allosteric drugs are currently receiving increased attention in drug discovery because drugs that target allosteric sites can provide important advantages over the corresponding orthosteric drugs including specific subtype selectivity within receptor families. Consequently, targeting allosteric sites, instead of orthosteric sites, can reduce drug-related side effects and toxicity. On the down side, allosteric drug discovery can be more challenging than traditional orthosteric drug discovery due to difficulties associated with determining the locations of allosteric sites and designing drugs based on these sites and the need for the allosteric effects to propagate through the structure, reach the ligand binding site and elicit a conformational change. In this study, we present computational tools ranging from the identification of potential allosteric sites to the design of "allosteric-like" modulator libraries. These tools may be particularly useful for allosteric drug discovery.

Design of a multiband near-infrared sky brightness monitor using an InSb detector.

PubMed

Dong, Shu-Cheng; Wang, Jian; Tang, Qi-Jie; Jiang, Feng-Xin; Chen, Jin-Ting; Zhang, Yi-Hao; Wang, Zhi-Yue; Chen, Jie; Zhang, Hong-Fei; Jiang, Hai-Jiao; Zhu, Qing-Feng; Jiang, Peng; Ji, Tuo

2018-02-01

Infrared sky background level is an important parameter of infrared astronomy observations from the ground, particularly for a candidate site of an infrared capable observatory since low background level is required for such a site. The Chinese astronomical community is looking for a suitable site for a future 12 m telescope, which is designed for working in both optical and infrared wavelengths. However, none of the proposed sites has been tested for infrared observations. Nevertheless, infrared sky background measurements are also important during the design of infrared observing instruments. Based on the requirement, in order to supplement the current site survey data and guide the design of future infrared instruments, a multiband near-infrared sky brightness monitor (MNISBM) based on an InSb sensor is designed in this paper. The MNISBM consists of an optical system, mechanical structure and control system, detector and cooler, high gain readout electronics, and operational software. It is completed and tested in the laboratory. The results show that the sensitivity of the MNISBM meets the requirements of the measurement of near-infrared sky background level of several well-known astronomical infrared observing sites.

A Bioinformatics Approach to the Identification of Variants Associated with Type 1 and Type 2 Diabetes Mellitus that Reside in Functionally Validated miRNAs Binding Sites.

PubMed

Ghaedi, Hamid; Bastami, Milad; Jahani, Mohammad Mehdi; Alipoor, Behnam; Tabasinezhad, Maryam; Ghaderi, Omar; Nariman-Saleh-Fam, Ziba; Mirfakhraie, Reza; Movafagh, Abolfazl; Omrani, Mir Davood; Masotti, Andrea

2016-06-01

The present work is aimed at finding variants associated with Type 1 and Type 2 diabetes mellitus (DM) that reside in functionally validated miRNAs binding sites and that can have a functional role in determining diabetes and related pathologies. Using bioinformatics analyses we obtained a database of validated polymorphic miRNA binding sites which has been intersected with genes related to DM or to variants associated and/or in linkage disequilibrium (LD) with it and is reported in genome-wide association studies (GWAS). The workflow we followed allowed us to find variants associated with DM that also reside in functional miRNA binding sites. These data have been demonstrated to have a functional role by impairing the functions of genes implicated in biological processes linked to DM. In conclusion, our work emphasized the importance of SNPs located in miRNA binding sites. The results discussed in this work may constitute the basis of further works aimed at finding functional candidates and variants affecting protein structure and function, transcription factor binding sites, and non-coding epigenetic variants, contributing to widen the knowledge about the pathogenesis of this important disease.

Design of a multiband near-infrared sky brightness monitor using an InSb detector

NASA Astrophysics Data System (ADS)

Dong, Shu-cheng; Wang, Jian; Tang, Qi-jie; Jiang, Feng-xin; Chen, Jin-ting; Zhang, Yi-hao; Wang, Zhi-yue; Chen, Jie; Zhang, Hong-fei; Jiang, Hai-jiao; Zhu, Qing-feng; Jiang, Peng; Ji, Tuo

2018-02-01

Infrared sky background level is an important parameter of infrared astronomy observations from the ground, particularly for a candidate site of an infrared capable observatory since low background level is required for such a site. The Chinese astronomical community is looking for a suitable site for a future 12 m telescope, which is designed for working in both optical and infrared wavelengths. However, none of the proposed sites has been tested for infrared observations. Nevertheless, infrared sky background measurements are also important during the design of infrared observing instruments. Based on the requirement, in order to supplement the current site survey data and guide the design of future infrared instruments, a multiband near-infrared sky brightness monitor (MNISBM) based on an InSb sensor is designed in this paper. The MNISBM consists of an optical system, mechanical structure and control system, detector and cooler, high gain readout electronics, and operational software. It is completed and tested in the laboratory. The results show that the sensitivity of the MNISBM meets the requirements of the measurement of near-infrared sky background level of several well-known astronomical infrared observing sites.

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure.

PubMed

Wulff, Philip; Thomas, Claudia; Sargent, Frank; Armstrong, Fraser A

2016-03-01

'Oxygen-tolerant' [NiFe]-hydrogenases can catalyze H2 oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O2 molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H2 oxidation released at the active site of one partner are immediately transferred to its counterpart when an O2 molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O2-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins.

CorA Is a Copper Repressible Surface-Associated Copper(I)-Binding Protein Produced in Methylomicrobium album BG8

PubMed Central

Johnson, Kenneth A.; Ve, Thomas; Larsen, Øivind; Pedersen, Rolf B.; Lillehaug, Johan R.; Jensen, Harald B.; Helland, Ronny; Karlsen, Odd A.

2014-01-01

CorA is a copper repressible protein previously identified in the methanotrophic bacterium Methylomicrobium album BG8. In this work, we demonstrate that CorA is located on the cell surface and binds one copper ion per protein molecule, which, based on X-ray Absorption Near Edge Structure analysis, is in the reduced state (Cu(I)). The structure of endogenously expressed CorA was solved using X-ray crystallography. The 1.6 Å three-dimensional structure confirmed the binding of copper and revealed that the copper atom was coordinated in a mononuclear binding site defined by two histidines, one water molecule, and the tryptophan metabolite, kynurenine. This arrangement of the copper-binding site is similar to that of its homologous protein MopE* from Metylococcus capsulatus Bath, confirming the importance of kynurenine for copper binding in these proteins. Our findings show that CorA has an overall fold similar to MopE, including the unique copper(I)-binding site and most of the secondary structure elements. We suggest that CorA plays a role in the M. album BG8 copper acquisition. PMID:24498370

Microbial Community Structure in a Serpentine-Hosted Abiotic Gas Seepage at the Chimaera Ophiolite, Turkey

PubMed Central

Sun, Li; Müller, Bettina; Ivarsson, Magnus; Hosgörmez, Hakan; Özcan, Dogacan; Broman, Curt; Schnürer, Anna

2017-01-01

ABSTRACT The surface waters at the ultramafic ophiolitic outcrop in Chimaera, Turkey, are characterized by high pH values and high metal levels due to the percolation of fluids through areas of active serpentinization. We describe the influence of the liquid chemistry, mineralogy, and H2 and CH4 levels on the bacterial community structure in a semidry, exposed, ultramafic environment. The bacterial and archaeal community structures were monitored using Illumina sequencing targeting the 16S rRNA gene. At all sampling points, four phyla, Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria, accounted for the majority of taxa. Members of the Chloroflexi phylum dominated low-diversity sites, whereas Proteobacteria dominated high-diversity sites. Methane, nitrogen, iron, and hydrogen oxidizers were detected as well as archaea and metal-resistant bacteria. IMPORTANCE Our study is a comprehensive microbial investigation of the Chimaera ophiolite. DNA has been extracted from 16 sites in the area and has been studied from microbial and geochemical points of view. We describe a microbial community structure that is dependent on terrestrial, serpentinization-driven abiotic H2, which is poorly studied due to the rarity of these environments on Earth. PMID:28389534

Microbial Community Structure in a Serpentine-Hosted Abiotic Gas Seepage at the Chimaera Ophiolite, Turkey.

PubMed

Neubeck, Anna; Sun, Li; Müller, Bettina; Ivarsson, Magnus; Hosgörmez, Hakan; Özcan, Dogacan; Broman, Curt; Schnürer, Anna

2017-06-15

The surface waters at the ultramafic ophiolitic outcrop in Chimaera, Turkey, are characterized by high pH values and high metal levels due to the percolation of fluids through areas of active serpentinization. We describe the influence of the liquid chemistry, mineralogy, and H 2 and CH 4 levels on the bacterial community structure in a semidry, exposed, ultramafic environment. The bacterial and archaeal community structures were monitored using Illumina sequencing targeting the 16S rRNA gene. At all sampling points, four phyla, Proteobacteria , Actinobacteria , Chloroflexi , and Acidobacteria , accounted for the majority of taxa. Members of the Chloroflexi phylum dominated low-diversity sites, whereas Proteobacteria dominated high-diversity sites. Methane, nitrogen, iron, and hydrogen oxidizers were detected as well as archaea and metal-resistant bacteria. IMPORTANCE Our study is a comprehensive microbial investigation of the Chimaera ophiolite. DNA has been extracted from 16 sites in the area and has been studied from microbial and geochemical points of view. We describe a microbial community structure that is dependent on terrestrial, serpentinization-driven abiotic H 2 , which is poorly studied due to the rarity of these environments on Earth. Copyright © 2017 Neubeck et al.

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bera, Asim K.; Aukema, Kelly G.; Elias, Mikael

Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas themore » two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel.« less

Application of Inter-Simple Sequence Repeat Markers in the Analysis of Populations of the Chagas Disease Vector Triatoma infestans (Hemiptera, Reduviidae)

PubMed Central

Pérez de Rosas, Alicia R.; Restelli, María F.; Fernández, Cintia J.; Blariza, María J.; García, Beatriz A.

2017-01-01

Here we apply inter-simple sequence repeat (ISSR) markers to explore the fine-scale genetic structure and dispersal in populations of Triatoma infestans. Five selected primers from 30 primers were used to amplify ISSRs by polymerase chain reaction. A total of 90 polymorphic bands were detected across 134 individuals captured from 11 peridomestic sites from the locality of San Martín (Capayán Department, Catamarca Province, Argentina). Significant levels of genetic differentiation suggest limited gene flow among sampling sites. Spatial autocorrelation analysis confirms that dispersal occurs on the scale of ∼469 m, suggesting that insecticide spraying should be extended at least within a radius of ∼500 m around the infested area. Moreover, Bayesian clustering algorithms indicated genetic exchange among different sites analyzed, supporting the hypothesis of an important role of peridomestic structures in the process of reinfestation. PMID:28115670

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

Epitope mapping of the domains of human angiotensin converting enzyme.

PubMed

Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

2006-06-01

Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi

2008-04-02

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) againstmore » hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.« less

Structural Characterization of the E2 Domain of APL-1, a C. Elegans Homolog of Human Amyloid Precursor Protein, and its Heparin Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoopes, J.; Liu, X; Xu, X

2010-01-01

The amyloid {beta}-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able tomore » map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.« less

Metal–organic and covalent organic frameworks as single-site catalysts

PubMed Central

Rogge, S. M. J.; Bavykina, A.; Hajek, J.; Garcia, H.; Olivos-Suarez, A. I.; Sepúlveda-Escribano, A.; Vimont, A.; Clet, G.; Bazin, P.; Kapteijn, F.

2017-01-01

Heterogeneous single-site catalysts consist of isolated, well-defined, active sites that are spatially separated in a given solid and, ideally, structurally identical. In this review, the potential of metal–organic frameworks (MOFs) and covalent organic frameworks (COFs) as platforms for the development of heterogeneous single-site catalysts is reviewed thoroughly. In the first part of this article, synthetic strategies and progress in the implementation of such sites in these two classes of materials are discussed. Because these solids are excellent playgrounds to allow a better understanding of catalytic functions, we highlight the most important recent advances in the modelling and spectroscopic characterization of single-site catalysts based on these materials. Finally, we discuss the potential of MOFs as materials in which several single-site catalytic functions can be combined within one framework along with their potential as powerful enzyme-mimicking materials. The review is wrapped up with our personal vision on future research directions. PMID:28338128

Conformational Sampling and Binding Site Assessment of Suppression of Tumorigenicity 2 Ectodomain

PubMed Central

Yang, Chao-Yie; Delproposto, James; Chinnaswamy, Krishnapriya; Brown, William Clay; Wang, Shuying; Stuckey, Jeanne A.; Wang, Xinquan

2016-01-01

Suppression of Tumorigenicity 2 (ST2), a member of the interleukin-1 receptor (IL-1R) family, activates type 2 immune responses to pathogens and tissue damage via binding to IL-33. Dysregulated responses contribute to asthma, graft-versus-host and autoinflammatory diseases and disorders. To study ST2 structure for inhibitor development, we performed the principal component (PC) analysis on the crystal structures of IL1-1R1, IL1-1R2, ST2 and the refined ST2 ectodomain (ST2ECD) models, constructed from previously reported small-angle X-ray scattering data. The analysis facilitates mapping of the ST2ECD conformations to PC subspace for characterizing structural changes. Extensive coverage of ST2ECD conformations was then obtained using the accelerated molecular dynamics simulations started with the IL-33 bound ST2ECD structure as instructed by their projected locations on the PC subspace. Cluster analysis of all conformations further determined representative conformations of ST2ECD ensemble in solution. Alignment of the representative conformations with the ST2/IL-33 structure showed that the D3 domain of ST2ECD (containing D1-D3 domains) in most conformations exhibits no clashes with IL-33 in the crystal structure. Our experimental binding data informed that the D1-D2 domain of ST2ECD contributes predominantly to the interaction between ST2ECD and IL-33 underscoring the importance of the D1-D2 domain in binding. Computational binding site assessment revealed one third of the total detected binding sites in the representative conformations may be suitable for binding to potent small molecules. Locations of these sites include the D1-D2 domain ST2ECD and modulation sites conformed to ST2ECD conformations. Our study provides structural models and analyses of ST2ECD that could be useful for inhibitor discovery. PMID:26735493

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

PubMed

Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

2014-03-14

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

Urban population genetics of slum-dwelling rats (Rattus norvegicus) in Salvador, Brazil

PubMed Central

Kajdacsi, Brittney; Costa, Federico; Hyseni, Chaz; Porter, Fleur; Brown, Julia; Rodrigues, Gorete; Farias, Helena; Reis, Mitermeyer G.; Childs, James E.; Ko, Albert I.; Caccone, Adalgisa

2013-01-01

Throughout the developing world, urban centers with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers, and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus), are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure, and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from 9 sites in the city of Salvador, Brazil. These sites were divided between three neighborhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographic distances. Most FST comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighborhoods or valleys within neighborhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies. PMID:24118116

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

The structural basis of secondary active transport mechanisms.

PubMed

Forrest, Lucy R; Krämer, Reinhard; Ziegler, Christine

2011-02-01

Secondary active transporters couple the free energy of the electrochemical potential of one solute to the transmembrane movement of another. As a basic mechanistic explanation for their transport function the model of alternating access was put forward more than 40 years ago, and has been supported by numerous kinetic, biochemical and biophysical studies. According to this model, the transporter exposes its substrate binding site(s) to one side of the membrane or the other during transport catalysis, requiring a substantial conformational change of the carrier protein. In the light of recent structural data for a number of secondary transport proteins, we analyze the model of alternating access in more detail, and correlate it with specific structural and chemical properties of the transporters, such as their assignment to different functional states in the catalytic cycle of the respective transporter, the definition of substrate binding sites, the type of movement of the central part of the carrier harboring the substrate binding site, as well as the impact of symmetry on fold-specific conformational changes. Besides mediating the transmembrane movement of solutes, the mechanism of secondary carriers inherently involves a mechanistic coupling of substrate flux to the electrochemical potential of co-substrate ions or solutes. Mainly because of limitations in resolution of available transporter structures, this important aspect of secondary transport cannot yet be substantiated by structural data to the same extent as the conformational change aspect. We summarize the concepts of coupling in secondary transport and discuss them in the context of the available evidence for ion binding to specific sites and the impact of the ions on the conformational state of the carrier protein, which together lead to mechanistic models for coupling. Copyright © 2010 Elsevier B.V. All rights reserved.

Structural basis of divergent cyclin-dependent kinase activation by Spy1/RINGO proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

McGrath, Denise A.; Fifield, Bre‐Anne; Marceau, Aimee H.

Cyclin-dependent kinases (Cdks) are principal drivers of cell division and are an important therapeutic target to inhibit aberrant proliferation. Cdk enzymatic activity is tightly controlled through cyclin interactions, posttranslational modifications, and binding of inhibitors such as the p27 tumor suppressor protein. Spy1/RINGO (Spy1) proteins bind and activate Cdk but are resistant to canonical regulatory mechanisms that establish cell-cycle checkpoints. Cancer cells exploit Spy1 to stimulate proliferation through inappropriate activation of Cdks, yet the mechanism is unknown. We have determined crystal structures of the Cdk2-Spy1 and p27-Cdk2-Spy1 complexes that reveal how Spy1 activates Cdk. We find that Spy1 confers structural changesmore » to Cdk2 that obviate the requirement of Cdk activation loop phosphorylation. Spy1 lacks the cyclin-binding site that mediates p27 and substrate affinity, explaining why Cdk-Spy1 is poorly inhibited by p27 and lacks specificity for substrates with cyclin-docking sites. We identify mutations in Spy1 that ablate its ability to activate Cdk2 and to proliferate cells. Our structural description of Spy1 provides important mechanistic insights that may be utilized for targeting upregulated Spy1 in cancer.« less

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

DOE PAGES

Laino, Aldana; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.; ...

2017-09-11

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus ( PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, andmore » it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant ( K m) was 1.7 mM with a k cat of 75 s –1. Two crystal structures are presented, the apo PvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Lastly, these results contribute to knowledge of mechanistic details of the function of arginine kinase.« less

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Laino, Aldana; Lopez-Zavala, Alonso A.; Garcia-Orozco, Karina D.

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus ( PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, andmore » it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant ( K m) was 1.7 mM with a k cat of 75 s –1. Two crystal structures are presented, the apo PvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Lastly, these results contribute to knowledge of mechanistic details of the function of arginine kinase.« less

A strand graph semantics for DNA-based computation

PubMed Central

Petersen, Rasmus L.; Lakin, Matthew R.; Phillips, Andrew

2015-01-01

DNA nanotechnology is a promising approach for engineering computation at the nanoscale, with potential applications in biofabrication and intelligent nanomedicine. DNA strand displacement is a general strategy for implementing a broad range of nanoscale computations, including any computation that can be expressed as a chemical reaction network. Modelling and analysis of DNA strand displacement systems is an important part of the design process, prior to experimental realisation. As experimental techniques improve, it is important for modelling languages to keep pace with the complexity of structures that can be realised experimentally. In this paper we present a process calculus for modelling DNA strand displacement computations involving rich secondary structures, including DNA branches and loops. We prove that our calculus is also sufficiently expressive to model previous work on non-branching structures, and propose a mapping from our calculus to a canonical strand graph representation, in which vertices represent DNA strands, ordered sites represent domains, and edges between sites represent bonds between domains. We define interactions between strands by means of strand graph rewriting, and prove the correspondence between the process calculus and strand graph behaviours. Finally, we propose a mapping from strand graphs to an efficient implementation, which we use to perform modelling and simulation of DNA strand displacement systems with rich secondary structure. PMID:27293306

Redox-linked Conformational Dynamics in Apoptosis Inducing Factor

PubMed Central

Sevrioukova, Irina F.

2009-01-01

Apoptosis inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH2-NAD charge-transfer complexes (CTC) proposed to be functionally important. To get a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the x-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) π-stacking interactions between coplanar nicotinamide, isoalloxazine and Phe309 rings, (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site, and (iii) an extensive hydrogen-bonding network involving His453, a key residue undergoing a conformational switch to directly interact and orient the nicotinamide in position optimal for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with the biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH. PMID:19447115

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

The m1A(58) modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI.

PubMed

Dégut, Clément; Ponchon, Luc; Folly-Klan, Marcia; Barraud, Pierre; Tisné, Carine

2016-03-01

The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Benthic Community Structure and Sediment Geochemical Properties at Hydrocarbon Seeps Along the Continental Slope of the Western North Atlantic

NASA Astrophysics Data System (ADS)

Demopoulos, A. W.; Bourque, J. R.; Brooke, S.

2015-12-01

Hydrocarbon seeps support distinct benthic communities capable of utilizing reduced chemical compounds for nutrition. In recent years, methane seepage has been increasingly documented along the continental slope of the U.S. Atlantic margin. In 2012 and 2013, two seeps were investigated in this region: a shallow site near Baltimore Canyon (410-450 m) and a deep site near Norfolk Canyon (1600 m). Both sites contain extensive mussel beds and microbial mats. Sediment cores and grab samples were collected to quantify the abundance, diversity, and community structure of benthic macrofauna (>300 mm) in relationship to the associated sediment environment (organic carbon and nitrogen, stable isotopes 13C and 15N, grain size, and depth) of mussel beds, mats, and slope habitats. Macrofaunal densities in microbial mats were four times greater than those present in mussel beds and slope sediments. Macrofaunal communities were distinctly different both between depths and among habitat types. Specifically, microbial mat sediments were dominated by the annelid families Dorvilleidae, Capitellidae, and Tubificidae, while mussel habitats had higher proportions of crustaceans. Diversity was lower in Baltimore microbial mat habitats, but higher in mussel and slope sediments compared to Norfolk seep habitats found at deeper depths. Multivariate statistical analysis identified sediment carbon:nitrogen (C:N) ratios and 13C values as important variables for structuring the macrofaunal communities. Higher C:N ratios were present within microbial mat habitats and depleted 13C values occurred in sediments adjacent to mussel beds found in Norfolk Canyon seeps. Differences in the quality and source of organic matter present in the seep habitats are known to be important drivers in macrofaunal community structure and associated food webs. The multivariate analysis provides new insight into the relative importance of the seep sediment quality in supporting dense macrofaunal communities compared to other seeps found throughout the region.

Crystal Structure of the Potassium Importing KdpFABC Membrane Complex

PubMed Central

Huang, Ching-Shin; Pedersen, Bjørn Panyella; Stokes, David Lloyd

2017-01-01

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four subunit potassium pump that maintains intracellular homeostasis as well as cell shape and turgor under conditions where potassium is limiting1. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the Superfamily of Potassium Transporters and another pump-like subunit (KdpB) belonging to the Superfamily of P-type ATPases. Although there is considerable structural and functional information about members from both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA as well as a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. Based on these observations, we propose an unprecedented mechanism that repurposes protein channel architecture for active transport across biomembranes. PMID:28636601

Thermodynamic and structural insights into CSL-DNA complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedmann, David R.; Kovall, Rhett A.

The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated bymore » a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo.« less

Structural charge site influence on the interlayer hydration of expandable three-sheet clay minerals

USGS Publications Warehouse

Kerns, Raymond L.; Mankin, Charles J.

1968-01-01

Previous investigations have demonstrated the influences of interlayer cation composition, relative humidity, temperature, and magnitude of interlayer surface charge on the interlayer hydration of montmorillonites and vermiculites. It has been suggested that the sites of layer charge deficiencies may also have an influence upon the amount of hydration that can take place in the interlayers of expandable clay minerals. If the interlayer cation-to-layer bonds are considered as ideally electrostatic, the magnitude of the forces resisting expansion may be expressed as a form of Coulomb's law. If this effect is significant, expandable structures in which the charge-deficiency sites are predominantly in the tetrahedral sheet should have less pronounced swelling properties than should structures possessing charge deficiencies located primarily in the octahedral sheet.Three samples that differed in location of layer charge sites were selected for study. An important selection criterion was a non-correlation between tetrahedral charge sites and high surface-charge density, and between octahedral charge sites and low surface-charge density.The effects of differences in interlayer cation composition were eliminated by saturating portions of each sample with the same cations. Equilibrium (001) d values at controlled constant humidities were used as a measure of the relative degree of interlayer hydration.Although no correlation could be made between the degree of interlayer hydration and total surface-charge density, the investigation does not eliminate total surface-charge density as being significant to the swelling properties of three-sheet clay-mineral structures. The results do indicate a correlation between more intense expandability and predominance of charge deficiencies in the octahedral sheet. Conversely, less intense swelling behavior is associated with predominantly tetrahedral charge deficiencies.

Ecological correlates of the non-indigenous parasitoid assemblage associated with a Hawaiian endemic moth.

PubMed

Kaufman, Leyla V; Wright, Mark G

2011-08-01

Understanding what ecological factors might predispose indigenous habitats to invasion by invasive species is an important aspect of conservation and invasive species management, particularly when biological control is considered for suppression of the invasive species. This study seeks to identify ecological factors that might play a role in determining the structure of the parasitoid assemblage associated with caterpillars of the endemic Hawaiian moth Udea stellata (Crambidae). Parasitoids were reared from field-collected U. stellata larvae at 18 locations. Fourteen environmental variables were measured at each site. Two multivariate analyses, principal component analysis (PCA) and partial redundancy analysis (RDA), were used to analyze the parasitoid assemblage across a range of habitats varying in environmental characteristics. The PCA analysis showed that the occurrence of some species were highly correlated, and associated with less disturbed sites, whereas other species were associated with sites of medium and high levels of disturbance. The RDA analysis showed that only three of the measured environmental variables (U. stellata density, elevation, and level of habitat disturbance) significantly explained variability in the parasitoid assemblage among sites. There was greater parasitoid species richness associated with U. stellata larvae at higher elevation sites with a lower degree of habitat disturbance by exotic vegetation. The purposely introduced parasitoid species were associated with the non-target moth at sites located at higher elevations with low levels of disturbance. Multivariate analysis has the potential to provide valuable insights into the identification of important environmental factors that mediate parasitoid assemblage structure and level of parasitism on a particular target or non-target species, and therefore facilitate identification of suitable target habitats or susceptible non-target habitats.

Mutagenesis Studies of the H5 Influenza Hemagglutinin Stem Loop Region*

PubMed Central

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L.; Mishra, Rama K.; Rong, Lijun; Caffrey, Michael

2014-01-01

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. PMID:24947513

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

PubMed

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L; Mishra, Rama K; Rong, Lijun; Caffrey, Michael

2014-08-08

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

454 pyrosequencing analysis of bacterial diversity revealed by a comparative study of soils from mining subsidence and reclamation areas.

PubMed

Li, Yuanyuan; Chen, Longqian; Wen, Hongyu; Zhou, Tianjian; Zhang, Ting; Gao, Xiali

2014-03-28

Significant alteration in the microbial community can occur across reclamation areas suffering subsidence from mining. A reclamation site undergoing fertilization practices and an adjacent coal-excavated subsidence site (sites A and B, respectively) were examined to characterize the bacterial diversity using 454 high-throughput 16S rDNA sequencing. The dominant taxonomic groups in both the sites were Proteobacteria, Acidobacteria, Bacteroidetes, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, and Firmicutes. However, the bacterial communities' abundance, diversity, and composition differed significantly between the sites. Site A presented higher bacterial diversity and more complex community structures than site B. The majority of sequences related to Proteobacteria, Gemmatimonadetes, Chloroflexi, Nitrospirae, Firmicutes, Betaproteobacteria, Deltaproteobacteria, and Anaerolineae were from site A; whereas those related to Actinobacteria, Planctomycetes, Bacteroidetes, Verrucomicrobia, Gammaproteobacteria, Nitriliruptoria, Alphaproteobacteria, and Phycisphaerae originated from site B. The distribution of some bacterial groups and subgroups in the two sites correlated with soil properties and vegetation due to reclamation practice. Site A exhibited enriched bacterial community, soil organic matter (SOM), and total nitrogen (TN), suggesting the presence of relatively diverse microorganisms. SOM and TN were important factors shaping the underlying microbial communities. Furthermore, the specific plant functional group (legumes) was also an important factor influencing soil microbial community composition. Thus, the effectiveness of 454 pyrosequencing in analyzing soil bacterial diversity was validated and an association between land ecological system restoration, mostly mediated by microbial communities, and an improvement in soil properties in coalmining reclamation areas was suggested.

Incorporating metals into de novo proteins.

PubMed

Peacock, Anna F A

2013-12-01

The de novo design of artificial metalloproteins from first-principles is a powerful strategy with which to establish the minimum structure required for function, as well as to identify the important design features for tuning the chemistry of the coordinated metal ion. Herein we describe recent contributions to this field, covering metallo-porphyrin, mononuclear and multinuclear metal ion sites engineered into de novo proteins. Using miniature artificial scaffolds these examples demonstrate that complex natural protein folds are not required to mimic naturally occurring metal ion sites in proteins. More importantly progress is being made to engineer de novo metalloproteins capable of performing functions not in the repertoire of biology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Functional identification and characterization of sodium binding sites in Na symporters

PubMed Central

Loo, Donald D. F.; Jiang, Xuan; Gorraitz, Edurne; Hirayama, Bruce A.; Wright, Ernest M.

2013-01-01

Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na+ in binding sites is beyond the resolution of the structures, two Na+ binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na+ binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na+ and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na+ binding, and that Na+ binding to Na2 promotes binding to Na1 and also sugar binding. PMID:24191006

Hierarchical drivers of reef-fish metacommunity structure.

PubMed

MacNeil, M Aaron; Graham, Nicholas A J; Polunin, Nicholas V C; Kulbicki, Michel; Galzin, René; Harmelin-Vivien, Mireille; Rushton, Steven P

2009-01-01

Coral reefs are highly complex ecological systems, where multiple processes interact across scales in space and time to create assemblages of exceptionally high biodiversity. Despite the increasing frequency of hierarchically structured sampling programs used in coral-reef science, little progress has been made in quantifying the relative importance of processes operating across multiple scales. The vast majority of reef studies are conducted, or at least analyzed, at a single spatial scale, ignoring the implicitly hierarchical structure of the overall system in favor of small-scale experiments or large-scale observations. Here we demonstrate how alpha (mean local number of species), beta diversity (degree of species dissimilarity among local sites), and gamma diversity (overall species richness) vary with spatial scale, and using a hierarchical, information-theoretic approach, we evaluate the relative importance of site-, reef-, and atoll-level processes driving the fish metacommunity structure among 10 atolls in French Polynesia. Process-based models, representing well-established hypotheses about drivers of reef-fish community structure, were assembled into a candidate set of 12 hierarchical linear models. Variation in fish abundance, biomass, and species richness were unevenly distributed among transect, reef, and atoll levels, establishing the relative contribution of variation at these spatial scales to the structure of the metacommunity. Reef-fish biomass, species richness, and the abundance of most functional-groups corresponded primarily with transect-level habitat diversity and atoll-lagoon size, whereas detritivore and grazer abundances were largely correlated with potential covariates of larval dispersal. Our findings show that (1) within-transect and among-atoll factors primarily drive the relationship between alpha and gamma diversity in this reef-fish metacommunity; (2) habitat is the primary correlate with reef-fish metacommunity structure at multiple spatial scales; and (3) inter-atoll connectedness was poorly correlated with the nonrandom clustering of reef-fish species. These results demonstrate the importance of modeling hierarchical data and processes in understanding reef-fish metacommunity structure.

Mutational analysis of microbial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) towards enhancement of binding affinity: A computational approach.

PubMed

Kumar, Pravin; Ghosh Sachan, Shashwati; Poddar, Raju

2017-10-01

Improving the industrial enzyme for better yield of the product is important and a challenging task. One of such important industrial enzymes is microbial Hydroxycinnamoyl-CoA hydratase-lyase (HCHL). It converts feruloyl-CoA to vanillin. We place our efforts towards the improvement of its catalytic activity with comprehensive computational investigation. Catalytic core of the HCHL was explored with molecular modeling and docking approaches. Site-directed mutations were introduced in the catalytic site of HCHL in a sequential manner to generate different mutants of HCHL. Basis of mutation is to increase the interaction between HCHL and substrate feruloyl-CoA through interatomic forces and hydrogen bond formation. A rigorous molecular dynamics (MD) simulation was performed to check the stability of mutant's structure. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), dynamic cross correlation (DCCM) and principal component analysis (PCA) were also performed to analyze flexibility and stability of structures. Docking studies were carried out between different mutants of HCHL and feruloyl-CoA. Investigation of the different binding sites and the interactions with mutant HCHLs and substrate allowed us to highlight the improved performance of mutants than wild type HCHL. This was further validated with MD simulation of complex consisting of different mutants and substrate. It further confirms all the structures are stable. However, mutant-2 showed better affinity towards substrate by forming hydrogen bond between active site and feruloyl-CoA. We propose that increase in hydrogen bond formation might facilitate in dissociation of vanillin from feruloyl-CoA. The current work may be useful for the future development of 'tailor-made' enzymes for better yield of vanillin. Copyright © 2017 Elsevier Inc. All rights reserved.

Plasticity of the Binding Site of Renin: Optimized Selection of Protein Structures for Ensemble Docking.

PubMed

Strecker, Claas; Meyer, Bernd

2018-05-29

Protein flexibility poses a major challenge to docking of potential ligands in that the binding site can adopt different shapes. Docking algorithms usually keep the protein rigid and only allow the ligand to be treated as flexible. However, a wrong assessment of the shape of the binding pocket can prevent a ligand from adapting a correct pose. Ensemble docking is a simple yet promising method to solve this problem: Ligands are docked into multiple structures, and the results are subsequently merged. Selection of protein structures is a significant factor for this approach. In this work we perform a comprehensive and comparative study evaluating the impact of structure selection on ensemble docking. We perform ensemble docking with several crystal structures and with structures derived from molecular dynamics simulations of renin, an attractive target for antihypertensive drugs. Here, 500 ns of MD simulations revealed binding site shapes not found in any available crystal structure. We evaluate the importance of structure selection for ensemble docking by comparing binding pose prediction, ability to rank actives above nonactives (screening utility), and scoring accuracy. As a result, for ensemble definition k-means clustering appears to be better suited than hierarchical clustering with average linkage. The best performing ensemble consists of four crystal structures and is able to reproduce the native ligand poses better than any individual crystal structure. Moreover this ensemble outperforms 88% of all individual crystal structures in terms of screening utility as well as scoring accuracy. Similarly, ensembles of MD-derived structures perform on average better than 75% of any individual crystal structure in terms of scoring accuracy at all inspected ensembles sizes.

Leaving Group Ability Observably Affects Transition State Structure in a Single Enzyme Active Site.

PubMed

Roston, Daniel; Demapan, Darren; Cui, Qiang

2016-06-15

A reaction's transition state (TS) structure plays a critical role in determining reactivity and has important implications for the design of catalysts, drugs, and other applications. Here, we explore TS structure in the enzyme alkaline phosphatase using hybrid Quantum Mechanics/Molecular Mechanics simulations. We find that minor perturbations to the substrate have major effects on TS structure and the way the enzyme stabilizes the TS. Substrates with good leaving groups (LGs) have little cleavage of the phosphorus-LG bond at the TS, while substrates with poor LGs have substantial cleavage of that bond. The results predict nonlinear free energy relationships for a single rate-determining step, and substantial differences in kinetic isotope effects for different substrates; both trends were observed in previous experimental studies, although the original interpretations differed from the present model. Moreover, due to different degrees of phosphorus-LG bond cleavage at the TS for different substrates, the LG is stabilized by different interactions at the TS: while a poor LG is directly stabilized by an active site zinc ion, a good LG is mainly stabilized by active site water molecules. Our results demonstrate the considerable plasticity of TS structure and stabilization in enzymes. Furthermore, perturbations to reactivity that probe TS structure experimentally (i.e., substituent effects) may substantially perturb the TS they aim to probe, and thus classical experimental approaches such as free energy relations should be interpreted with care.

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

PubMed Central

Qin, Ling; Hiser, Carrie; Mulichak, Anne; Garavito, R. Michael; Ferguson-Miller, Shelagh

2006-01-01

Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 Å resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His–Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins. PMID:17050688

ENANTIOMER FRACTIONS OF CHLORDANE COMPOUNDS IN SEDIMENT SAMPLES FROM U.S. GEOLOGICAL SURVEY SITES IN LAKES, RIVERS, AND RESERVOIRS

EPA Science Inventory

More than 500 important environmental contaminants are chiral (having structures that are nonsuperimposible mirror images). Although enantiomer pairs have identical physical-chemical properties, their toxicity, biodegradation, and environmental fate often are different. Cyclo...

Molecular modeling and docking studies of human 5-hydroxytryptamine 2A (5-HT2A) receptor for the identification of hotspots for ligand binding.

PubMed

Kanagarajadurai, Karuppiah; Malini, Manoharan; Bhattacharya, Aditi; Panicker, Mitradas M; Sowdhamini, Ramanathan

2009-12-01

The serotonergic system has been implicated in emotional and cognitive function. In particular, 5-HT(2A) (5-hydroxytrytamine receptor 2A) is attributed to a number of disorders like schizophrenia, depression, eating disorders and anxiety. 5-HT(2A), being a GPCR (G-protein coupled receptor), is important in the pharmaceutical industry as a proven target for these disorders. Despite their extensive clinical importance, the structural studies of this protein is lacking due to difficulties in determining its crystal structure. We have performed sequence analysis and molecular modeling of 5-HT(2A) that has revealed a set of conserved residues and motifs considered to play an important role in maintaining structural integrity and function of the receptor. The analysis also revealed a set of residues specific to the receptor which distinguishes them from other members of the subclass and their orthologs. Further, starting from the model structure of human 5-HT(2A) receptor, docking studies were attempted to envisage how it might interact with eight of its ligands (such as serotonin, dopamine, DOI, LSD, haloperidol, ketanserin, risperidone and clozapine). The binding studies of dopamine to 5-HT(2A) receptor can bring up better understanding in the etiology of a number of neurological disorders involving both these two receptors. Our sequence analysis and study of interactions of this receptor with other ligands reveal additional residue hotspots such as Asn 363 and Tyr 370. The function of these residues can be further analyzed by rational design of site-directed mutagenesis. Two distinct binding sites are identified which could play important roles in ligand binding and signaling.

Exploring protein structure and dynamics through a project-oriented biochemistry laboratory module.

PubMed

Lipchock, James M; Ginther, Patrick S; Douglas, Bonnie B; Bird, Kelly E; Patrick Loria, J

2017-09-01

Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017. © 2017 The International Union of Biochemistry and Molecular Biology.

Food-web structure of seagrass communities across different spatial scales and human impacts.

PubMed

Coll, Marta; Schmidt, Allison; Romanuk, Tamara; Lotze, Heike K

2011-01-01

Seagrass beds provide important habitat for a wide range of marine species but are threatened by multiple human impacts in coastal waters. Although seagrass communities have been well-studied in the field, a quantification of their food-web structure and functioning, and how these change across space and human impacts has been lacking. Motivated by extensive field surveys and literature information, we analyzed the structural features of food webs associated with Zostera marina across 16 study sites in 3 provinces in Atlantic Canada. Our goals were to (i) quantify differences in food-web structure across local and regional scales and human impacts, (ii) assess the robustness of seagrass webs to simulated species loss, and (iii) compare food-web structure in temperate Atlantic seagrass beds with those of other aquatic ecosystems. We constructed individual food webs for each study site and cumulative webs for each province and the entire region based on presence/absence of species, and calculated 16 structural properties for each web. Our results indicate that food-web structure was similar among low impact sites across regions. With increasing human impacts associated with eutrophication, however, food-web structure show evidence of degradation as indicated by fewer trophic groups, lower maximum trophic level of the highest top predator, fewer trophic links connecting top to basal species, higher fractions of herbivores and intermediate consumers, and higher number of prey per species. These structural changes translate into functional changes with impacted sites being less robust to simulated species loss. Temperate Atlantic seagrass webs are similar to a tropical seagrass web, yet differed from other aquatic webs, suggesting consistent food-web characteristics across seagrass ecosystems in different regions. Our study illustrates that food-web structure and functioning of seagrass habitats change with human impacts and that the spatial scale of food-web analysis is critical for determining results.

Food-Web Structure of Seagrass Communities across Different Spatial Scales and Human Impacts

PubMed Central

Coll, Marta; Schmidt, Allison; Romanuk, Tamara; Lotze, Heike K.

2011-01-01

Seagrass beds provide important habitat for a wide range of marine species but are threatened by multiple human impacts in coastal waters. Although seagrass communities have been well-studied in the field, a quantification of their food-web structure and functioning, and how these change across space and human impacts has been lacking. Motivated by extensive field surveys and literature information, we analyzed the structural features of food webs associated with Zostera marina across 16 study sites in 3 provinces in Atlantic Canada. Our goals were to (i) quantify differences in food-web structure across local and regional scales and human impacts, (ii) assess the robustness of seagrass webs to simulated species loss, and (iii) compare food-web structure in temperate Atlantic seagrass beds with those of other aquatic ecosystems. We constructed individual food webs for each study site and cumulative webs for each province and the entire region based on presence/absence of species, and calculated 16 structural properties for each web. Our results indicate that food-web structure was similar among low impact sites across regions. With increasing human impacts associated with eutrophication, however, food-web structure show evidence of degradation as indicated by fewer trophic groups, lower maximum trophic level of the highest top predator, fewer trophic links connecting top to basal species, higher fractions of herbivores and intermediate consumers, and higher number of prey per species. These structural changes translate into functional changes with impacted sites being less robust to simulated species loss. Temperate Atlantic seagrass webs are similar to a tropical seagrass web, yet differed from other aquatic webs, suggesting consistent food-web characteristics across seagrass ecosystems in different regions. Our study illustrates that food-web structure and functioning of seagrass habitats change with human impacts and that the spatial scale of food-web analysis is critical for determining results. PMID:21811637

Analysis of factors influencing hydration site prediction based on molecular dynamics simulations.

PubMed

Yang, Ying; Hu, Bingjie; Lill, Markus A

2014-10-27

Water contributes significantly to the binding of small molecules to proteins in biochemical systems. Molecular dynamics (MD) simulation based programs such as WaterMap and WATsite have been used to probe the locations and thermodynamic properties of hydration sites at the surface or in the binding site of proteins generating important information for structure-based drug design. However, questions associated with the influence of the simulation protocol on hydration site analysis remain. In this study, we use WATsite to investigate the influence of factors such as simulation length and variations in initial protein conformations on hydration site prediction. We find that 4 ns MD simulation is appropriate to obtain a reliable prediction of the locations and thermodynamic properties of hydration sites. In addition, hydration site prediction can be largely affected by the initial protein conformations used for MD simulations. Here, we provide a first quantification of this effect and further indicate that similar conformations of binding site residues (RMSD < 0.5 Å) are required to obtain consistent hydration site predictions.

Analysis of sequencing data for probing RNA secondary structures and protein-RNA binding in studying posttranscriptional regulations.

PubMed

Hu, Xihao; Wu, Yang; Lu, Zhi John; Yip, Kevin Y

2016-11-01

High-throughput sequencing has been used to study posttranscriptional regulations, where the identification of protein-RNA binding is a major and fast-developing sub-area, which is in turn benefited by the sequencing methods for whole-transcriptome probing of RNA secondary structures. In the study of RNA secondary structures using high-throughput sequencing, bases are modified or cleaved according to their structural features, which alter the resulting composition of sequencing reads. In the study of protein-RNA binding, methods have been proposed to immuno-precipitate (IP) protein-bound RNA transcripts in vitro or in vivo By sequencing these transcripts, the protein-RNA interactions and the binding locations can be identified. For both types of data, read counts are affected by a combination of confounding factors, including expression levels of transcripts, sequence biases, mapping errors and the probing or IP efficiency of the experimental protocols. Careful processing of the sequencing data and proper extraction of important features are fundamentally important to a successful analysis. Here we review and compare different experimental methods for probing RNA secondary structures and binding sites of RNA-binding proteins (RBPs), and the computational methods proposed for analyzing the corresponding sequencing data. We suggest how these two types of data should be integrated to study the structural properties of RBP binding sites as a systematic way to better understand posttranscriptional regulations. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

An in-silico insight into the characteristics of β-propeller phytase.

PubMed

Mathew, Akash; Verma, Anukriti; Gaur, Smriti

2014-06-01

Phytase is an enzyme that is found extensively in the plant kingdom and in some species of bacteria and fungi. This paper identifies and analyses the available full length sequences of β-propeller phytases (BPP). BPP was chosen due to its potential applicability in the field of aquaculture. The sequences were obtained from the Uniprot database and subject to various online bioinformatics tools to elucidate the physio-chemical characteristics, secondary structures and active site compositions of BPP. Protparam and SOPMA were used to analyse the physiochemical and secondary structure characteristics, while the Expasy online modelling tool and CASTp were used to model the 3-D structure and identify the active sites of the BPP sequences. The amino acid compositions of the four sequences were compared and composed in a graphical format to identify similarities and highlight the potentially important amino acids that form the active site of BPP. This study aims to analyse BPP and contribute to the clarification of the molecular mechanism involved in the enzyme activity of BPP and contribute in part to the possibility of constructing a synthetic version of BPP.

Detection of Shallow Buried Archaeological Remains Structure using 2-D Resistivity Method at Sungai Batu, Lembah Bujang, Kedah

NASA Astrophysics Data System (ADS)

Ismail, Nurina; Nordiana, M. M.; Saidin, M.; Masnan, S. S. K.; Abir, I. A.

2018-04-01

In an archaeological study, 2-D resistivity becomes important tools for the scientific investigation and also can be applied to map shallow subsurface structures at the archaeological site. The main purpose of the research is to locate the shallow buried archaeological remains structure in the area of Sungai Batu, Lembah Bujang, Kedah (Malaysia). Resistivity surveys have been applied using a Pole-dipole array with both 5 survey lines for SB1 and SB2 sites using 0.75 m electrode spacing. Both results revealed high resistivity zone with ≥ 3000 Ωm at a depth ranging from 0-1.5 m at the study area. SB1 site conducting an excavation to validate the result obtain while SB2 is not conducting any excavation. Besides that, the other characteristics of the buried archaeological remains are indicated as mound area and there is exposed remain found on top of the surface. Based on the archaeological evidence, the higher resistivity value indicates the interesting anomaly which is clay bricks for archaeological excavation.

Structures of closed and open conformations of dimeric human ATM

PubMed Central

Baretić, Domagoj; Pollard, Hannah K.; Fisher, David I.; Johnson, Christopher M.; Santhanam, Balaji; Truman, Caroline M.; Kouba, Tomas; Fersht, Alan R.; Phillips, Christopher; Williams, Roger L.

2017-01-01

ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase–related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate–binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATM structure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation. PMID:28508083

Structural aspects of denitrifying enzymes.

PubMed

Moura, I; Moura, J J

2001-04-01

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

Structural insights into the human RyR2 N-terminal region involved in cardiac arrhythmias

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borko, Ľubomír; Bauerová-Hlinková, Vladena, E-mail: vladena.bauerova@savba.sk; Hostinová, Eva

2014-11-01

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix. Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardiomyocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminusmore » is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals C{sup α} atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.« less

Structural analysis of the endogenous glycoallergen Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis and its recognition by human basophils

PubMed Central

Rodríguez-Romero, Adela; Hernández-Santoyo, Alejandra; Fuentes-Silva, Deyanira; Palomares, Laura A.; Muñoz-Cruz, Samira; Yépez-Mulia, Lilian; Orozco-Martínez, Socorro

2014-01-01

Endogenous glycosylated Hev b 2 (endo-β-1,3-glucanase) from Hevea brasiliensis is an important latex allergen that is recognized by IgE antibodies from patients who suffer from latex allergy. The carbohydrate moieties of Hev b 2 constitute a potentially important IgE-binding epitope that could be responsible for its cross-reactivity. Here, the structure of the endogenous isoform II of Hev b 2 that exhibits three post-translational modifications, including an N-terminal pyro­glutamate and two glycosylation sites at Asn27 and at Asn314, is reported from two crystal polymorphs. These modifications form a patch on the surface of the molecule that is proposed to be one of the binding sites for IgE. A structure is also proposed for the most important N-glycan present in this protein as determined by digestion with specific enzymes. To analyze the role of the carbohydrate moieties in IgE antibody binding and in human basophil activation, the glycoallergen was enzymatically deglycosylated and evaluated. Time-lapse automated video microscopy of basophils stimulated with glycosylated Hev b 2 revealed basophil activation and degranulation. Immunological studies suggested that carbohydrates on Hev b 2 represent an allergenic IgE epitope. In addition, a dimer was found in each asymmetric unit that may reflect a regulatory mechanism of this plant defence protein. PMID:24531467

Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

PubMed Central

Powell, Amy J; Peabody, David S

2001-01-01

Background The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets. Results We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half. Conclusions These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4. PMID:11504563

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain*S⃞

PubMed Central

Bottomley, Matthew J.; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-01-01

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions. PMID:18614528

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

PubMed

Bottomley, Matthew J; Lo Surdo, Paola; Di Giovine, Paolo; Cirillo, Agostino; Scarpelli, Rita; Ferrigno, Federica; Jones, Philip; Neddermann, Petra; De Francesco, Raffaele; Steinkühler, Christian; Gallinari, Paola; Carfí, Andrea

2008-09-26

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

PubMed Central

Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

2017-01-01

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. DOI: http://dx.doi.org/10.7554/eLife.26640.001 PMID:28792888

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

PubMed

Zhu, Hui; Shuman, Stewart

2005-04-01

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Structure/function relationships in serotonin transporter: new insights from the structure of a bacterial transporter.

PubMed

Rudnick, G

2006-01-01

Serotonin transporter (SERT) serves the important function of taking up serotonin (5-HT) released during serotonergic neurotransmission. It is the target for important therapeutic drugs and psychostimulants. SERT catalyzes the influx of 5-HT together with Na+ and Cl- in a 1:1:1 stoichiometry. In the same catalytic cycle, there is coupled efflux of one K+ ion. SERT is one member of a large family of amino acid and amine transporters that is believed to utilize similar mechanisms of transport. A bacterial member of this family was recently crystallized, revealing the structural basis of these transporters. In light of the new structure, previous results with SERT have been re-interpreted, providing new insight into the substrate binding site, the permeation pathway, and the conformational changes that occur during the transport cycle.

Temporal change in biological community structure in the Fountain Creek basin, Colorado, 2001-2008

USGS Publications Warehouse

Zuellig, Robert E.; Bruce, James F.; Stogner, Sr., Robert W.

2010-01-01

In 2001, the U.S. Geological Survey, in cooperation with Colorado Springs City Engineering, began a study to better understand the relations between environmental characteristics and biological communities in the Fountain Creek basin in order to aide water-resource management and guide future monitoring activities. To accomplish this task, environmental (streamflow, habitat, and water chemistry) and biological (fish and macroinvertebrate) data were collected annually at 24 sites over a 6- or 8-year period (fish, 2003 to 2008; macroinvertebrates, 2001 to 2008). For this report, these data were first analyzed to determine the presence of temporal change in macroinvertebrate and fish community structure among years using nonparametric multivariate statistics. Where temporal change in the biological communities was found, these data were further analyzed using additional nonparametric multivariate techniques to determine which subset of selected streamflow, habitat, or water-chemistry variables best described site-specific changes in community structure relative to a gradient of urbanization. This study identified significant directional patterns of temporal change in macroinvertebrate and fish community structure at 15 of 24 sites in the Fountain Creek basin. At four of these sites, changes in environmental variables were significantly correlated with the concurrent temporal change identified in macroinvertebrate and fish community structure (Monument Creek above Woodmen Road at Colorado Springs, Colo.; Monument Creek at Bijou Street at Colorado Springs, Colo.; Bear Creek near Colorado Springs, Colo.; Fountain Creek at Security, Colo.). Combinations of environmental variables describing directional temporal change in the biota appeared to be site specific as no single variable dominated the results; however, substrate composition variables (percent substrate composition composed of sand, gravel, or cobble) collectively were present in 80 percent of the environmental variable subsets that were significantly correlated with temporal change in the macroinvertebrate and fish community structure. Other important environmental variables related to temporal change in the biological community structure included those describing channel form (streambank height) and streamflow (normalized annual mean daily flow, high flood-pulse count). Site-specific results from this study were derived from a relatively small number of observations (6 or 8 years of data); therefore, additional years of data may reveal other sites with temporal change in biological community structure, or could define stronger and more consistent linkages between environmental variables and observed temporal change. Likewise current variable subsets could become weaker. Nonetheless, there were several sites where temporal change was detected in this study that could not be explained by the available environmental variables studied herein. Modification of current data-collection activities may be necessary to better understand site-specific temporal relations between biological communities and environmental variables.

MDC1: The art of keeping things in focus.

PubMed

Jungmichel, Stephanie; Stucki, Manuel

2010-08-01

The chromatin structure is important for recognition and repair of DNA damage. Many DNA damage response proteins accumulate in large chromatin domains flanking sites of DNA double-strand breaks. The assembly of these structures-usually termed DNA damage foci-is primarily regulated by MDC1, a large nuclear mediator/adaptor protein that is composed of several distinct structural and functional domains. Here, we are summarizing the latest discoveries about the mechanisms by which MDC1 mediates DNA damage foci formation, and we are reviewing the considerable efforts taken to understand the functional implication of these structures.

Electron microscopy study of gold nanoparticles deposited on transition metal oxides.

PubMed

Akita, Tomoki; Kohyama, Masanori; Haruta, Masatake

2013-08-20

Many researchers have investigated the catalytic performance of gold nanoparticles (GNPs) supported on metal oxides for various catalytic reactions of industrial importance. These studies have consistently shown that the catalytic activity and selectivity depend on the size of GNPs, the kind of metal oxide supports, and the gold/metal oxide interface structure. Although researchers have proposed several structural models for the catalytically active sites and have identified the specific electronic structures of GNPs induced by the quantum effect, recent experimental and theoretical studies indicate that the perimeter around GNPs in contact with the metal oxide supports acts as an active site in many reactions. Thus, it is of immense importance to investigate the detailed structures of the perimeters and the contact interfaces of gold/metal oxide systems by using electron microscopy at an atomic scale. This Account describes our investigation, at the atomic scale using electron microscopy, of GNPs deposited on metal oxides. In particular, high-resolution transmission electron microscopy (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) are valuable tools to observe local atomic structures, as has been successfully demonstrated for various nanoparticles, surfaces, and material interfaces. TEM can be applied to real powder catalysts as received without making special specimens, in contrast to what is typically necessary to observe bulk materials. For precise structure analyses at an atomic scale, model catalysts prepared by using well-defined single-crystalline substrates are also adopted for TEM observations. Moreover, aberration-corrected TEM, which has high spatial resolution under 0.1 nm, is a promising tool to observe the interface structure between GNPs and metal oxide supports including oxygen atoms at the interfaces. The oxygen atoms in particular play an important role in the behavior of gold/metal oxide interfaces, because they may participate in catalytic reaction steps. Detailed information about the interfacial structures between GNPs and metal oxides provides valuable structure models for theoretical calculations which can elucidate the local electronic structure effective for activating a reactant molecule. Based on our observations with HRTEM and HAADF-STEM, we report the detailed structure of gold/metal oxide interfaces.

The importance of hydrology in restoration of bottomland hardwood wetland functions

USGS Publications Warehouse

Hunter, R.G.; Faulkner, S.P.; Gibson, K.A.

2008-01-01

Bottomland hardwood (BLH) forests have important biogeochemical functions and it is well known that certain structural components, including pulsed hydrology, hydric soils, and hydrophytic vegetation, enhance these functions. It is unclear, however, how functions of restored BLH wetlands compare to mature, undisturbed wetlands. We measured a suite of structural and functional attributes in replicated natural BLH wetlands (NAT), restored BLH wetlands with hydrology re-established (RWH), and restored BLH wetlands without hydrology re-established (RWOH) in this study. Trees were replanted in all restored wetlands at least four years prior to the study and those wetlands with hydrology re-established had flashboard risers placed in drainage ditches to allow seasonal surface flooding. Vegetation, soils, and selected biogeochemical functions were characterized at each site. There was a marked difference in woody vegetation among the wetlands that was due primarily to site age. There was also a difference in herbaceous vegetation among the restored sites that may have been related to differences in age or hydrology. Water table fluctuations of the RWH wetlands were comparable to those of the NAT wetlands. Thus, placing flashboard risers in existing drainage ditches, along with proper management, can produce a hydroperiod that is similar to that of a relatively undisturbed BLH. Average length of saturation within the upper 15 cm of soils was 37, 104, and 97 days for RWOH, RWH, and NAT, respectively. Soil moisture, denitrification potential, and soluble organic carbon concentrations differed among wetland sites, but soil carbon and nitrogen concentrations, heterotrophic microbial activity, and readily mineralizable carbon concentrations did not. Significant linear relationships were also found between soil moisture and heterotrophic microbial activity, readily mineralizable carbon, and soluble organic carbon. In addition, sedimentation rates were higher in NAT and RWH wetlands than in RWOH sites. Results of this study suggest that reconnection of bottomland hardwood wetlands to their surrounding watershed through the restoration of surface hydrology is necessary to restore wetland functions important to nutrient and sediment removal. ?? 2008 The Society of Wetland Scientists.

Sodium and Potassium Ions in Proteins and Enzyme Catalysis.

PubMed

Vašák, Milan; Schnabl, Joachim

2016-01-01

The group I alkali metal ions Na(+) and K(+) are ubiquitous components of biological fluids that surround biological macromolecules. They play important roles other than being nonspecific ionic buffering agents or mediators of solute exchange and transport. Molecular evolution and regulated high intracellular and extracellular M(+) concentrations led to incorporation of selective Na(+) and K(+) binding sites into enzymes to stabilize catalytic intermediates or to provide optimal positioning of substrates. The mechanism of M(+) activation, as derived from kinetic studies along with structural analysis, has led to the classification of cofactor-like (type I) or allosteric effector (type II) activated enzymes. In the type I mechanism substrate anchoring to the enzyme active site is mediated by M(+), often acting in tandem with a divalent cation like Mg(2+), Mn(2+) or Zn(2+). In the allosteric type II mechanism, M(+) binding enhances enzyme activity through conformational transitions triggered upon binding to a distant site. In this chapter, following the discussion of the coordination chemistry of Na(+) and K(+) ions and the structural features responsible for the metal binding site selectivity in M(+)-activated enzymes, well-defined examples of M(+)-activated enzymes are used to illustrate the structural basis for type I and type II activation by Na(+) and K(+).

Mapping intermolecular interactions and active site conformations: from human MMP-1 crystal structure to molecular dynamics free energy calculations.

PubMed

Nash, Anthony; Birch, Helen L; de Leeuw, Nora H

2017-02-01

The zinc-dependent Matrix Metalloproteinases (MMPs) found within the extracellular matrix (ECM) of vertebrates are linked to pathological processes such as arthritis, skin ulceration and cancer. Although a general backbone proteolytic mechanism is understood, crystallographic data continue to suggest an active site that is too narrow to encompass the respective substrate. We present a fully parameterised Molecular Dynamics (MD) study of the structural properties of an MMP-1-collagen crystallographic structure (Protein Data Bank - 4AUO), followed by an exploration of the free energy surface of a collagen polypeptide chain entering the active site, using a combined meta-dynamics and umbrella sampling (MDUS) approach. We conclude that the interactions between MMP-1 and the collagen substrate are in good agreement with a number of experimental studies. As such, our unrestrained MD simulations and our MDUS results, which indicate an energetic barrier for a local uncoiling and insertion event, can inform future investigations of the collagen-peptide non-bonded association steps with the active site prior to proteolytic mechanisms. The elucidation of such free energy barriers provides a better understanding of the role of the enzyme in the ECM and is important in the design of future MMP inhibitors.

Differences in microbial community structure and nitrogen cycling in natural and drained tropical peatland soils.

PubMed

Espenberg, Mikk; Truu, Marika; Mander, Ülo; Kasak, Kuno; Nõlvak, Hiie; Ligi, Teele; Oopkaup, Kristjan; Maddison, Martin; Truu, Jaak

2018-03-16

Tropical peatlands, which play a crucial role in the maintenance of different ecosystem services, are increasingly drained for agriculture, forestry, peat extraction and human settlement purposes. The present study investigated the differences between natural and drained sites of a tropical peatland in the community structure of soil bacteria and archaea and their potential to perform nitrogen transformation processes. The results indicate significant dissimilarities in the structure of soil bacterial and archaeal communities as well as nirK, nirS, nosZ, nifH and archaeal amoA gene-possessing microbial communities. The reduced denitrification and N 2 -fixing potential was detected in the drained tropical peatland soil. In undisturbed peatland soil, the N 2 O emission was primarily related to nirS-type denitrifiers and dissimilatory nitrate reduction to ammonium, while the conversion of N 2 O to N 2 was controlled by microbes possessing nosZ clade I genes. The denitrifying microbial community of the drained site differed significantly from the natural site community. The main reducers of N 2 O were microbes harbouring nosZ clade II genes in the drained site. Additionally, the importance of DNRA process as one of the controlling mechanisms of N 2 O fluxes in the natural peatlands of the tropics revealed from the results of the study.

Influence of land-use legacies following shrub reduction and seeding of big sagebrush sites

USDA-ARS?s Scientific Manuscript database

Big sagebrush (Artemisia tridentata Nutt.) plant communities provide important economic and ecosystem values, but often require management to reduce shrub density and rehabilitate understory vegetation. We studied vegetation structure and plant community responses to a two-way chain harrow treatment...

Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies.

PubMed

Meola, Annalisa; Tarr, Alexander W; England, Patrick; Meredith, Luke W; McClure, C Patrick; Foung, Steven K H; McKeating, Jane A; Ball, Jonathan K; Rey, Felix A; Krey, Thomas

2015-02-01

Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic region contributes to the evasion of the humoral host immune response, facilitating chronicity and the viral spread of HCV within an infected individual. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of kin structure among Guam rail founders: a comparison of pedigrees and DNA profiles

USGS Publications Warehouse

Haig, Susan M.; Ballou, J.D.; Casna, N.J.

1994-01-01

Kin structure among founders can have a significant effect on subsequent population structure. Here we use the correlation between DNA profile similarity and relatedness calculated from pedigrees to test hypotheses regarding kin structure among founders to the captive Guam rail (Rallus owstoni) population. Five different pedigrees were generated under the following hypotheses: (i) founders are unrelated; (ii) founders are unrelated except for same-nest chicks; (iii) founders from the same major site are siblings; (iv) founders from the same local site are siblings; and (v) founders are related as defined by a UPGMA cluster analysis of DNA similarity data. Relatedness values from pedigrees 1, 2 and 5 had the highest correlation with DNA similarity but the correlation between relatedness and similarity were not significantly different among pedigrees. Pedigree 5 resulted in the highest correlation overall when using only relatedness values that changed as a result of different founder hypotheses. Thus, founders were assigned relatedness based on pedigree 5 because it had the highest correlations with DNA similarity, was the most conservative approach, and incorporated all field data. The analyses indicated that estimating relatedness using DNA profiles remains problematic, therefore we compared mean kinship, a measure of genetic importance, with mean DNA profile similarity to determine if genetic importance among individuals could be determined via use of DNA profiles alone. The significant correlation suggests this method may provide more information about population structure than was previously thought. Thus, DNA profiles can provide a reasonable explanation for founder relatedness and mean DNA profile similarity may be helpful in determining relative genetic importance of individuals when detailed pedigrees are absent.

Relative Importance of Biotic and Abiotic Forces on the Composition and Dynamics of a Soft-Sediment Intertidal Community

PubMed Central

Barbeau, Myriam A.

2016-01-01

Top-down, bottom-up, middle-out and abiotic factors are usually viewed as main forces structuring biological communities, although assessment of their relative importance, in a single study, is rarely done. We quantified, using multivariate methods, associations between abiotic and biotic (top-down, bottom-up and middle-out) variables and infaunal population/community variation on intertidal mudflats in the Bay of Fundy, Canada, over two years. Our analysis indicated that spatial structural factors like site and plot accounted for most of the community and population variation. Although we observed a significant relationship between the community/populations and the biotic and abiotic variables, most were of minor importance relative to the structural factors. We suggest that community and population structure were relatively uncoupled from the structuring influences of biotic and abiotic factors in this system because of high concentrations of resources that sustain high densities of infauna and limit exploitative competition. Furthermore, we hypothesize that the infaunal community primarily reflects stochastic spatial events, namely a “first come, first served” process. PMID:26790098

Clonality and diversity of the fish pathogen Lactococcus garvieae in Mediterranean countries.

PubMed

Eyngor, Marina; Zlotkin, Amir; Ghittino, Claudio; Prearo, Marino; Douet, Diane-Gaëlle; Chilmonczyk, Stefan; Eldar, Avi

2004-09-01

Infection with Lactococcus garvieae is considered the most important risk factor for the European trout industry, and the losses are approximately 50% of the total production. To improve our understanding of the genetic links among strains originating from different countries, we examined the population structure of L. garvieae by comparing 81 strains isolated from different sources and ecosystems (41 farms in six countries) in which the bacterium is commonly found. Genetic similarities (as assessed with molecular tools, including restriction fragment length polymorphism ribotyping with two endonucleases) were compared with serological data. The combined results reveal that in endemic sites the bacterial population displays a clonal structure, whereas bacterial diversity characterizes sites where the infection is sporadic.

When small changes matter: the role of cross-scale interactions between habitat and ecological connectivity in recovery.

PubMed

Thrush, Simon F; Hewitt, Judi E; Lohrer, Andrew M; Chiaroni, Luca D

2013-01-01

Interaction between the diversity of local communities and the degree of connectivity between them has the potential to influence local recovery rates and thus profoundly affect community dynamics in the face of the cumulative impacts that occur across regions. Although such complex interactions have been modeled, field experiments in natural ecosystems to investigate the importance of interactions between local and regional processes are rare, especially so in coastal marine seafloor habitats subjected to many types of disturbance. We conducted a defaunation experiment at eight subtidal sites, incorporating manipulation of habitat structure, to test the relative importance of local habitat features and colonist supply in influencing macrobenthic community recovery rate. Our sites varied in community composition, habitat characteristics, and hydrodynamic conditions, and we conducted the experiment in two phases, exposing defaunated plots to colonists during periods of either high or low larval colonist supply. In both phases of the experiment, five months after disturbance, we were able to develop models that explained a large proportion of variation in community recovery rate between sites. Our results emphasize that the connectivity to the regional species pool influences recovery rate, and although local habitat effects were important, the strength of these effects was affected by broader-scale site characteristics and connectivity. Empirical evidence that cross-scale interactions are important in disturbance-recovery dynamics emphasizes the complex dynamics underlying seafloor community responses to cumulative disturbance.

Insights from a network of long-term measurements of biosphere-atmospheric exchanges of water vapor and carbon dioxide in a water-limited semiarid region

NASA Astrophysics Data System (ADS)

Scott, Russell; Biederman, Joel

2017-04-01

Around one-third of Earth's land surface is classified as semiarid, and carbon dioxide exchange in these regions has been shown to be an important regulator of both the trend and interannual variability of the terrestrial carbon sink. Fifteen years ago, when we began making measurements of biosphere-atmospheric exchanges of energy, water vapor, and carbon dioxide using eddy covariance in southern Arizona USA, there was paucity of semiarid observations in flux networks like AmeriFlux and EuroFlux. We started by establishing riparian sites across a woody plant encroachment gradient to quantify the productivity and consumptive plant water use along a iconic and ecologically important desert river. Soon thereafter, we added semiarid grassland, shrubland, and savanna sites that do not have access to groundwater in order to better understand how water limitation and changes in vegetation structure affect ecosystem productivity. Here, we highlight the value of multiyear, multisite flux data for addressing regional to global scale problems associated with groundwater pumping, land cover change, drought, and climate change. For the riparian sites, we find that ecosystem water availability is altered by vegetation structure such that ecosystems with more deeply rooted trees have higher productivity but at a cost of greater groundwater use. For the non-riparian sites, precipitation strongly controls ecosystem water availability and the resultant productivity, but differences in ecosystem structure impact water use efficiency due to the partitioning of evapotranspiration into its component sources. Also, the productivity at sites with more grass, and less woody, plants responds more quickly to precipitation fluctuations including long-term drought conditions. In semiarid regions, variability in water and carbon fluxes is much larger than in more mesic climes. Across our riparian and non-riparian sites, access to more stable groundwater reserves reduces variability in water and carbon fluxes and can decouple ecosystem productivity from precipitation. Finally, we show that the 8% increase in carbon dioxide concentrations over the period of our measurements may be altering ecosystem water use efficiency, a result that is expected and has been reported for northeastern US forests.

Insights from a network of long-term measurements of biosphere-atmospheric exchanges of water vapor and carbon dioxide in southern Arizona

NASA Astrophysics Data System (ADS)

Scott, R. L.; Barron-Gafford, G.; Biederman, J. A.

2016-12-01

Around one-third of Earth's land surface is classified as semiarid, and carbon dioxide exchange in these regions has been shown to be an important regulator of both the trend and interannual variability of the terrestrial carbon sink. Fifteen years ago, when we began making measurements of biosphere-atmospheric exchanges of energy, water vapor, and carbon dioxide using eddy covariance in southern Arizona USA, there was paucity of semiarid observations in flux networks like Ameriflux. We started by establishing riparian sites across a woody plant encroachment gradient to quantify the productivity and consumptive plant water use along a iconic and ecologically important desert river. Soon thereafter, we added semiarid grassland, shrubland, and savanna sites that do not have access to groundwater in order to better understand how water limitation and changes in vegetation structure affect ecosystem productivity. Here, we highlight the value of multiyear, multisite flux data for addressing regional to global scale problems associated with groundwater pumping, land cover change, drought, and climate change. For the riparian sites, we find that ecosystem water availability is altered by vegetation structure such that ecosystems with more deeply rooted trees have higher productivity but at a cost of greater groundwater use. For the non-riparian sites, precipitation strongly controls ecosystem water availability and the resultant productivity, but differences in ecosystem structure impact water use efficiency due to the partitioning of evapotranspiration into its component sources. Also, the productivity at sites with more grass, and less woody, plants responds more quickly to precipitation fluctuations including long-term drought conditions. In semiarid regions, variability in water and carbon fluxes is much larger than in more mesic climes. Across our riparian and non-riparian sites, access to more stable groundwater reserves reduces variability in water and carbon fluxes and can decouple ecosystem productivity from precipitation. Finally, we show that the 8% increase in carbon dioxide concentrations over the period of our measurements has not resulted in any apparent changes in ecosystem water use efficiency that is expected and has been reported for northeastern US forests.

Substrate-bound outward-open state of the betaine transporter BetP provides insights into Na+ coupling

NASA Astrophysics Data System (ADS)

Perez, Camilo; Faust, Belinda; Mehdipour, Ahmad Reza; Francesconi, Kevin A.; Forrest, Lucy R.; Ziegler, Christine

2014-07-01

The Na+-coupled betaine symporter BetP shares a highly conserved fold with other sequence unrelated secondary transporters, for example, with neurotransmitter symporters. Recently, we obtained atomic structures of BetP in distinct conformational states, which elucidated parts of its alternating-access mechanism. Here, we report a structure of BetP in a new outward-open state in complex with an anomalous scattering substrate, adding a fundamental piece to an unprecedented set of structural snapshots for a secondary transporter. In combination with molecular dynamics simulations these structural data highlight important features of the sequential formation of the substrate and sodium-binding sites, in which coordinating water molecules play a crucial role. We observe a strictly interdependent binding of betaine and sodium ions during the coupling process. All three sites undergo progressive reshaping and dehydration during the alternating-access cycle, with the most optimal coordination of all substrates found in the closed state.

Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3).

PubMed

Mulepati, Sabin; Bailey, Scott

2011-09-09

RNA transcribed from clustered regularly interspaced short palindromic repeats (CRISPRs) protects many prokaryotes from invasion by foreign DNA such as viruses, conjugative plasmids, and transposable elements. Cas3 (CRISPR-associated protein 3) is essential for this CRISPR protection and is thought to mediate cleavage of the foreign DNA through its N-terminal histidine-aspartate (HD) domain. We report here the 1.8 Å crystal structure of the HD domain of Cas3 from Thermus thermophilus HB8. Structural and biochemical studies predict that this enzyme binds two metal ions at its active site. We also demonstrate that the single-stranded DNA endonuclease activity of this T. thermophilus domain is activated not by magnesium but by transition metal ions such as manganese and nickel. Structure-guided mutagenesis confirms the importance of the metal-binding residues for the nuclease activity and identifies other active site residues. Overall, these results provide a framework for understanding the role of Cas3 in the CRISPR system.

Structural Basis for Methyl Transfer by a Radical SAM Enzyme

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boal, Amie K.; Grove, Tyler L.; McLaughlin, Monica I.

2014-10-02

The radical S-adenosyl-l-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys{sup 355}) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys{sup 355} is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfermore » binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site.« less

Synthesis, QSAR, and Molecular Dynamics Simulation of Amidino-substituted Benzimidazoles as Dipeptidyl Peptidase III Inhibitors.

PubMed

Rastija, Vesna; Agić, Dejan; Tomiš, Sanja; Nikolič, Sonja; Hranjec, Marijana; Grace, Karminski-Zamola; Abramić, Marija

2015-01-01

A molecular modeling study is performed on series of benzimidazol-based inhibitors of human dipeptidyl peptidase III (DPP III). An eight novel compounds were synthesized in excellent yields using green chemistry approach. This study is aimed to elucidate the structural features of benzimidazole derivatives required for antagonism of human DPP III activity using Quantitative Structure-Activity Relationship (QSAR) analysis, and to understand the mechanism of one of the most potent inhibitor binding into the active site of this enzyme, by molecular dynamics (MD) simulations. The best model obtained includes S3K and RDF045m descriptors which have explained 89.4 % of inhibitory activity. Depicted moiety for strong inhibition activity matches to the structure of most potent compound. MD simulation has revealed importance of imidazolinyl and phenyl groups in the mechanism of binding into the active site of human DPP III.

Engineered control of enzyme structural dynamics and function.

PubMed

Boehr, David D; D'Amico, Rebecca N; O'Rourke, Kathleen F

2018-04-01

Enzymes undergo a range of internal motions from local, active site fluctuations to large-scale, global conformational changes. These motions are often important for enzyme function, including in ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post-translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus-responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine. © 2018 The Protein Society.

Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein and member of the highly conserved YjgF family

PubMed Central

Sinha, Sangita; Rappu, Pekka; Lange, S. C.; Mäntsälä, Pekka; Zalkin, Howard; Smith, Janet L.

1999-01-01

The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-Å crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c/YjgF/UK114 family of proteins. PMID:10557275

Understanding customers' intention to use social network sites as complaint channel: an analysis of young customers' perspectives

NASA Astrophysics Data System (ADS)

Setiawan, Retno Agus; Setyohadi, Djoko Budiyanto; Pranowo

2018-02-01

Social network sites (SNSs) have grown rapidly in recent years. More and more companies have used SNSs as part of their business strategy. SNSs offer numerous advantages, especially in enhancing communication. SNSs have a potential as a new complaint channel for young customers to file their complaints to companies. The objective of this study is to investigate the acceptance of SNSs as complaint channel based on TAM. A structured questionnaire was distributed to young participants, which collected 222 valid questionnaires. Furthermore, structural equation modeling was utilized to investigate the structural model. The results revealed that perceived ease of use and perceived usefulness have a positive correlation on the attitude towards SNSs. While the attitude plays an important role in understanding customers' intention to use SNSs to voice complaints. However perceived usefulness has no significant impact on intention to use. Limitations and further research were also discussed.

Crystal structure of human nicotinamide riboside kinase.

PubMed

Khan, Javed A; Xiang, Song; Tong, Liang

2007-08-01

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

Planned residential units: New development trajectories

NASA Astrophysics Data System (ADS)

Fedchenko, Irina

2017-01-01

The paper summarizes the transformation patterns of functional, morphological, social, and administrative structures of planned residential units - district (Russia, Eastern Europe), neighborhood (USA), community (UK, Europe), as the smallest structural and planning elements of the settlements. The study is based on the author's own on-site survey of the existing and new planned residential units, as well as on the analysis of theoretical sources. The multidisciplinary analysis of the theoretical concepts and on-site survey results showed that planned residential units formed in the early twentieth century retain their social and planning importance and identity, evolve and acquire new features and forms. At the same time, according to the current regulatory and legal instruments they remain basic planning elements of urban structure in the early twenty-first century. This paper also includes experimental analysis of the theoretical concepts of planned residential units' transformation, their conceptual planning model and formation principles in the early twenty-first century.

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

PubMed

Krajewski, Wojciech W; Collins, Ruairi; Holmberg-Schiavone, Lovisa; Jones, T Alwyn; Karlberg, Tobias; Mowbray, Sherry L

2008-01-04

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

Visualizing Active-Site Dynamics in Single Crystals of HePTP: Opening of the WPD Loop Involves Coordinated Movement of the E Loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Critton; L Tautz; R Page

2011-12-31

Phosphotyrosine hydrolysis by protein tyrosine phosphatases (PTPs) involves substrate binding by the PTP loop and closure over the active site by the WPD loop. The E loop, located immediately adjacent to the PTP and WPD loops, is conserved among human PTPs in both sequence and structure, yet the role of this loop in substrate binding and catalysis is comparatively unexplored. Hematopoietic PTP (HePTP) is a member of the kinase interaction motif (KIM) PTP family. Compared to other PTPs, KIM-PTPs have E loops that are unique in both sequence and structure. In order to understand the role of the E loopmore » in the transition between the closed state and the open state of HePTP, we identified a novel crystal form of HePTP that allowed the closed-state-to-open-state transition to be observed within a single crystal form. These structures, which include the first structure of the HePTP open state, show that the WPD loop adopts an 'atypically open' conformation and, importantly, that ligands can be exchanged at the active site, which is critical for HePTP inhibitor development. These structures also show that tetrahedral oxyanions bind at a novel secondary site and function to coordinate the PTP, WPD, and E loops. Finally, using both structural and kinetic data, we reveal a novel role for E-loop residue Lys182 in enhancing HePTP catalytic activity through its interaction with Asp236 of the WPD loop, providing the first evidence for the coordinated dynamics of the WPD and E loops in the catalytic cycle, which, as we show, is relevant to multiple PTP families.« less

Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

PubMed

Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

2015-12-15

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site.

PubMed

Pang, Yu; Lan, Wanjun; Huang, Xuelei; Zuo, Guanke; Liu, Hui; Zhang, Jingyan

2015-10-01

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe(3+) ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe(3+) is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe(3+) ions, two residue clusters of OxOx were assigned as potential Fe(3+) binding sites, the mechanism of the inhibition of Fe(3+) was delineated. Importantly, the residues that interact with Fe(3+) ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe(3+) highlights insight into substrate binding site in OxOx.

A quantum chemical study for exploring the inhibitory effect of nitrogen containing species on the adsorption of polynuclear aromatic hydrocarbons over a Bronsted acid site

NASA Astrophysics Data System (ADS)

Celis-Cornejo, C. M.; Garnica Mantilla, M. M.; Baldovino-Medrano, V. G.; Ramírez-Caballero, G. E.

2016-08-01

The analysis of the inhibitory effect of nitrogenated compounds on the hydroprocessing and hydropurification of oil derived fuels is important to produce cleaner fuels. In this work, density functional theory calculations were performed to investigate the effect of the nitrogen containing molecules on the adsorption of Polynuclear Aromatic Hydrocarbons (PAHs). Mordenite was chosen as a zeolitic structure for simulating a Bronsted acid site. The character of the acid site was confirmed by both a vibrational frequency calculation and a Bader charge analysis. From the adsorption calculations, it was found that the adsorption energy of PAHs increases with the number of aromatic rings in the structure. Also, the nitrogen containing species possibly inhibit more extensively two and three rings PAHs because of their lower adsorption energies. Finally, it was observed that the nitrogen species tend to drag the proton from the mordenite acid site. This explains the inhibitory effect in the adsorption of PAHs and contributes to understanding the dynamics of hydrocarbon hydroprocessing in refineries.

Terrestrial laser scanning for heritage conservation: the Cologne Cathedral documentation project

NASA Astrophysics Data System (ADS)

Pritchard, D.; Sperner, J.; Hoepner, S.; Tenschert, R.

2017-08-01

Contemporary terrestrial laser scanners and photogrammetric imaging systems are an invaluable tool in providing objectively precise, as-built records of existing architectural, engineering and industrial sites. The comprehensive three-dimensional (3D) recording of culturally important sites such as heritage buildings, monuments, and sites can serve a variety of invaluable purposes; the data can assist in the conservation, management, and repair of a structure, as well as provide a visually engaging educational resource for both the public and scholars. The acquired data acts as a form of digital preservation, a timeless virtual representation of the as-built structure. The technical capability of these systems is particularly suited for the documentation of a richly articulated and detailed building such as the high Gothic Cologne Cathedral. The 3D documentation of the Cologne Cathedral UNESCO World Heritage Site is a multiphase project developed by Heriot-Watt University, Edinburgh in partnership with the Fresenius University of Applied Sciences, Cologne, and the Metropolitankapitel der Hohen Domkirche Köln Dombauhütte. The project has also received generous support from Zoller + Fröhlich (Z+F) and the City of Cologne.

Shared regulatory sites are abundant in the human genome and shed light on genome evolution and disease pleiotropy.

PubMed

Tong, Pin; Monahan, Jack; Prendergast, James G D

2017-03-01

Large-scale gene expression datasets are providing an increasing understanding of the location of cis-eQTLs in the human genome and their role in disease. However, little is currently known regarding the extent of regulatory site-sharing between genes. This is despite it having potentially wide-ranging implications, from the determination of the way in which genetic variants may shape multiple phenotypes to the understanding of the evolution of human gene order. By first identifying the location of non-redundant cis-eQTLs, we show that regulatory site-sharing is a relatively common phenomenon in the human genome, with over 10% of non-redundant regulatory variants linked to the expression of multiple nearby genes. We show that these shared, local regulatory sites are linked to high levels of chromatin looping between the regulatory sites and their associated genes. In addition, these co-regulated gene modules are found to be strongly conserved across mammalian species, suggesting that shared regulatory sites have played an important role in shaping human gene order. The association of these shared cis-eQTLs with multiple genes means they also appear to be unusually important in understanding the genetics of human phenotypes and pleiotropy, with shared regulatory sites more often linked to multiple human phenotypes than other regulatory variants. This study shows that regulatory site-sharing is likely an underappreciated aspect of gene regulation and has important implications for the understanding of various biological phenomena, including how the two and three dimensional structures of the genome have been shaped and the potential causes of disease pleiotropy outside coding regions.

Transverse zones controlling the structural evolution of the Zipaquira Anticline (Eastern Cordillera, Colombia): Regional implications

NASA Astrophysics Data System (ADS)

García, Helbert; Jiménez, Giovanny

2016-08-01

We report paleomagnetic, magnetic fabric and structural results from 21 sites collected in Cretaceous marine mudstones and Paleogene continental sandstones from the limbs, hinge and transverse zones of the Zipaquira Anticline (ZA). The ZA is an asymmetrical fold with one limb completely overturned by processes like gravity and salt tectonics, and marked by several axis curvatures. The ZA is controlled by at least two (2) transverse zones known as the Neusa and Zipaquira Transverse Zones (NTZ and ZTZ, respectively). Magnetic mineralogy methods were applied at different sites and the main carriers of the magnetic properties are paramagnetic components with some sites being controlled by hematite and magnetite. Magnetic fabric analysis shows rigid-body rotation for the back-limb in the ZA, while the forelimb is subjected to internal deformation. Structural and paleomagnetic data shows the influence of the NTZ and ZTZ in the evolution of the different structures like the ZA and the Zipaquira, Carupa, Rio Guandoque, Las Margaritas and Neusa faults, controlling several factors as vergence, extension, fold axis curvature and stratigraphic detatchment. Clockwise rotations unraveled a block segmentation following a discontinuos model caused by transverse zones and one site reported a counter clockwise rotation associated with a left-lateral strike slip component for transverse faults (e.g. the Neusa Fault). We propose that diverse transverse zones have been active since Paleogene times, playing an important role in the tectonic evolution of the Cundinamarca sub-basin and controlling the structural evolution of folds and faults with block segmentation and rotations.

Structure and thermal expansion of Ca9Gd(VO4)7: A combined powder-diffraction and dilatometric study of a Czochralski-grown crystal

NASA Astrophysics Data System (ADS)

Paszkowicz, Wojciech; Shekhovtsov, Alexei; Kosmyna, Miron; Loiko, Pavel; Vilejshikova, Elena; Minikayev, Roman; Romanowski, Przemysław; Wierzchowski, Wojciech; Wieteska, Krzysztof; Paulmann, Carsten; Bryleva, Ekaterina; Belikov, Konstantin; Fitch, Andrew

2017-11-01

Materials of the Ca9RE(VO4)7 (CRVO) formula (RE = rare earth) and whitlockite-related structures are considered for applications in optoelectronics, e.g., in white-light emitting diodes and lasers. In the CRVO structure, the RE atoms are known to share the site occupation with Ca atoms at two or three among four Ca sites, with partial occupancy values depending on the choice of the RE atom. In this work, the structure and quality of a Czochralski-grown crystal of this family, Ca9Gd(VO4)7 (CGVO), are studied using X-ray diffraction methods. The room-temperature structure is refined using the powder diffraction data collected at a high-resolution synchrotron beamline ID22 (ESRF, Grenoble); for comparison purposes, a laboratory diffraction pattern was collected and analyzed, as well. The site occupancies are discussed on the basis of comparison with literature data of isostructural synthetic crystals of the CRVO series. The results confirm the previously reported site-occupation scheme and indicate a tendency of the CGVO compound to adopt a Gd-deficient composition. Moreover, the thermal expansion coefficient is determined for CGVO as a function of temperature in the 302-1023 K range using laboratory diffraction data. Additionally, for CGVO and six other single crystals of the same family, thermal expansion is studied in the 298-473 K range, using the dilatometric data. The magnitude and anisotropy of thermal expansion, being of importance for laser applications, are discussed for these materials.

Structure-Based Alteration of Substrate Specificity and Catalytic Activity of Sulfite Oxidase from Sulfite Oxidation to Nitrate Reduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, James A.; Wilson, Heather L.; Rajagopalan, K.V.

Eukaryotic sulfite oxidase is a dimeric protein that contains the molybdenum cofactor and catalyzes the metabolically essential conversion of sulfite to sulfate as the terminal step in the metabolism of cysteine and methionine. Nitrate reductase is an evolutionarily related molybdoprotein in lower organisms that is essential for growth on nitrate. In this study, we describe human and chicken sulfite oxidase variants in which the active site has been modified to alter substrate specificity and activity from sulfite oxidation to nitrate reduction. On the basis of sequence alignments and the known crystal structure of chicken sulfite oxidase, two residues are conservedmore » in nitrate reductases that align with residues in the active site of sulfite oxidase. On the basis of the crystal structure of yeast nitrate reductase, both positions were mutated in human sulfite oxidase and chicken sulfite oxidase. The resulting double-mutant variants demonstrated a marked decrease in sulfite oxidase activity but gained nitrate reductase activity. An additional methionine residue in the active site was proposed to be important in nitrate catalysis, and therefore, the triple variant was also produced. The nitrate reducing ability of the human sulfite oxidase triple mutant was nearly 3-fold greater than that of the double mutant. To obtain detailed structural data for the active site of these variants, we introduced the analogous mutations into chicken sulfite oxidase to perform crystallographic analysis. The crystal structures of the Mo domains of the double and triple mutants were determined to 2.4 and 2.1 {angstrom} resolution, respectively.« less

Diverse Actions and Target-Site Selectivity of Neonicotinoids: Structural Insights

PubMed Central

Matsuda, Kazuhiko; Kanaoka, Satoshi; Akamatsu, Miki; Sattelle, David B.

2009-01-01

The nicotinic acetylcholine receptors (nAChRs) are targets for human and veterinary medicines as well as insecticides. Subtype-selectivity among the diverse nAChR family members is important for medicines targeting particular disorders, and pest-insect selectivity is essential for the development of safer, environmentally acceptable insecticides. Neonicotinoid insecticides selectively targeting insect nAChRs have important applications in crop protection and animal health. Members of this class exhibit strikingly diverse actions on their nAChR targets. Here we review the chemistry and diverse actions of neonicotinoids on insect and mammalian nAChRs. Electrophysiological studies on native nAChRs and on wild-type and mutagenized recombinant nAChRs have shown that basic residues particular to loop D of insect nAChRs are likely to interact electrostatically with the nitro group of neonicotinoids. In 2008, the crystal structures were published showing neonicotinoids docking into the acetylcholine binding site of molluscan acetylcholine binding proteins with homology to the ligand binding domain (LBD) of nAChRs. The crystal structures showed that 1) glutamine in loop D, corresponding to the basic residues of insect nAChRs, hydrogen bonds with the NO2 group of imidacloprid and 2) neonicotinoid-unique stacking and CH-π bonds at the LBD. A neonicotinoid-resistant strain obtained by laboratory-screening has been found to result from target site mutations, and possible reasons for this are also suggested by the crystal structures. The prospects of designing neonicotinoids that are safe not only for mammals but also for beneficial insects such as honey bees (Apis mellifera) are discussed in terms of interactions with non-α nAChR subunits. PMID:19321668

Life-history and habitat features influence the within-river genetic structure of Atlantic salmon.

PubMed

Vähä, Juha-Pekka; Erkinaro, Jaakko; Niemelä, Eero; Primmer, Craig R

2007-07-01

Defining populations and identifying ecological and life-history characteristics affecting genetic structure is important for understanding species biology and hence, for managing threatened or endangered species or populations. In this study, populations of the world's largest indigenous Atlantic salmon (Salmo salar) stock were first inferred using model-based clustering methods, following which life-history and habitat variables best predicting the genetic diversity of populations were identified. This study revealed that natal homing of Atlantic salmon within the Teno River system is accurate at least to the tributary level. Generally, defining populations by main tributaries was observed to be a reasonable approach in this large river system, whereas in the mainstem of the river, the number of inferred populations was fewer than the number of distinct sampling sites. Mainstem and headwater populations were genetically more diverse and less diverged, while each tributary fostered a distinct population with high genetic differentiation and lower genetic diversity. Population structure and variation in genetic diversity among populations were poorly explained by geographical distance. In contrast, age-structure, as estimated by the proportion of multisea-winter spawners, was the most predictive variable in explaining the variation in the genetic diversity of the populations. This observation, being in agreement with theoretical predictions, emphasizes the essence of large multisea-winter females in maintaining the genetic diversity of populations. In addition, the unique genetic diversity of populations, as estimated by private allele richness, was affected by the ease of accessibility of a site, with more difficult to access sites having lower unique genetic diversity. Our results show that despite this species' high capacity for migration, tributaries foster relatively closed populations with little gene flow which will be important to consider when developing management strategies for the system.

Molecular dynamics simulation studies and in vitro site directed mutagenesis of avian beta-defensin Apl_AvBD2

PubMed Central

2010-01-01

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions. PMID:20122244

[Rapid ecological assessment of tropical fish communities in a gold mine area of Costa Rica].

PubMed

Espinoza Mendiola, Mario

2008-12-01

Gold mining impacts have generated a great concern regarding aquatic systems and habitat fragmentation. Anthropogenic disturbances on the structure and heterogeneity of a system can have an important effect on aquatic community stability. Ecological rapid assessments (1996, 2002, and 2007) were employed to determine the structure, composition and distribution of tropical fish communities in several rivers and smaller creeks from a gold mining area in Cerro Crucitas, Costa Rica. In addition, species composition and relative abundance were related with habitat structure. A total of 35 species were registered, among which sardine Astyanax aeneus (Characidae) and livebearer Alfaro cultratus (Poeciliidae) were the most abundant fish (71%). The highest species richness was observed in Caño Crucitas (s=19) and Minas Creek (s=18). Significant differences in fish communities structure and composition from Infiernillo river and Minas creek were observed (lamda = 0.0, F(132, 66) = 2.24, p < 0.001). Presence and/or absence of certain species such as Dormitor gobiomorus, Rhamdia nicaraguensis, Parachromis loiseillei and Atractosteus tropicus explained most of the spatial variation among sites. Habitat structure also contributed to explain differences among sites (lamda = 0.004, F(60.183) = 5.52, p < 0.001). Substratum (soft and hard bottom types) and habitat attributes (elevation, width and depth) explained most of the variability observed in Infiernillo River, Caño Crucitas and Tamagá Creek. In addition, a significant association between fish species and habitat structure was observed. This study reveals a high complexity in tropical fish communities that inhabit a gold mine area. Furthermore, it highlights the importance of habitat heterogeneity in fish community dynamics. The loss and degradation of aquatic systems in Cerro Crucitas can have a strong negative effect on fish community structure and composition of local species. A better understanding of the use of specific habitats that serve as essential fish habitats can improve tropical fish conservation and management strategies, thus increasing local diversity, and thereby, the biological importance of the area.

Orange-red emitting Gd2Zr2O7:Sm3+: Structure-property correlation, optical properties and defect spectroscopy

NASA Astrophysics Data System (ADS)

Gupta, Santosh K.; Reghukumar, C.; Sudarshan, K.; Ghosh, P. S.; Pathak, Nimai; Kadam, R. M.

2018-05-01

Local structure analysis of dopant ion, understanding host to dopant energy transfer dynamics and defects characterization in a doped material which plays an important role in the designing a highly efficient opto-electronic material. In this connection a new Sm3+ doped Gd2Zr2O7 pyrochlore material was synthesized using gel-combustion technique and was characterized systematically using X-ray diffraction (XRD), time resolved photoluminescence spectroscopy (TRPLS), positron annihilation lifetime spectroscopy (PALS) and density functional theory (DFT) based ab-initio calculation. Based on DFT site selective energetics calculation and luminescence decay measurement, it was observed that the Sm3+ was distributed at both Gd3+ and Zr4+ site with higher Sm3+ fraction at the Gd3+ site. PALS was used to probe the presence of defects in the phosphor. In this work intense orange-red emission is realized through manipulating the energy transfer from host defect emission (oxygen vacancies) to Sm3+ which allows color emission from green in undoped to orange-red in doped samples. Effect of dopant concentration and annealing temperature was probed using TRPLS and PALS. These all information is highly important for researcher looking to achieve pyrochlore based phosphor materials with high quantum yield.

Microscopic origin of entropy-driven polymorphism in hybrid organic-inorganic perovskite materials

NASA Astrophysics Data System (ADS)

Butler, Keith T.; Svane, Katrine; Kieslich, Gregor; Cheetham, Anthony K.; Walsh, Aron

2016-11-01

Entropy is a critical, but often overlooked, factor in determining the relative stabilities of crystal phases. The importance of entropy is most pronounced in softer materials, where small changes in free energy can drive phase transitions, which has recently been demonstrated in the case of organic-inorganic hybrid-formate perovskites. In this Rapid Communication we demonstrate the interplay between composition and crystal structure that is responsible for the particularly pronounced role of entropy in determining polymorphism in hybrid organic-inorganic materials. Using ab initio based lattice dynamics, we probe the origins and effects of vibrational entropy of four archetype perovskite (A B X3 ) structures. We consider an inorganic material (SrTiO3), an A -site hybrid-halide material (CH3NH3) PbI3 , a X -site hybrid material KSr (BH4)3 , and a mixed A - and X -site hybrid-formate material (N2H5) Zn (HCO2)3 , comparing the differences in entropy between two common polymorphs. The results demonstrate the importance of low-frequency intermolecular modes in determining the phase stability in these materials. The understanding gained allows us to propose a general principle for the relative stability of different polymorphs of hybrid materials as temperature is increased.

Riparian vegetation structure and the hunting behavior of adult estuarine crocodiles.

PubMed

Evans, Luke J; Davies, Andrew B; Goossens, Benoit; Asner, Gregory P

2017-01-01

Riparian ecosystems are amongst the most biodiverse tropical habitats. They are important, and essential, ecological corridors, linking remnant forest fragments. In this study, we hypothesised that crocodile's actively select nocturnal resting locations based on increased macaque predation potential. We examined the importance of riparian vegetation structure in the maintenance of crocodile hunting behaviours. Using airborne Light Detection and Ranging (LiDAR) and GPS telemetry on animal movement, we identified the repeated use of nocturnal resting sites by adult estuarine crocodiles (Crocodylus porosus) throughout the fragmented Lower Kinabatangan Wildlife Sanctuary in Sabah, Malaysia. Crocodile resting locations were found to resemble, in terms of habitat characteristics, the sleeping sites of long-tailed macaque; positioned in an attempt to avoid predation by terrestrial predators. We found individual crocodiles were actively selecting overhanging vegetation and that the protrusion of trees from the tree line was key to site selection by crocodiles, as well as influencing both the presence and group size of sleeping macaques. Although these findings are correlational, they have broad management implications, with the suggestion that riparian corridor maintenance and quality can have implications beyond that of terrestrial fauna. We further place our findings in the context of the wider ecosystem and the maintenance of trophic interactions, and discuss how future habitat management has the potential to mitigate human-wildlife conflict.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Helix A Stabilization Precedes Amino-terminal Lobe Activation upon Calcium Binding to Calmodulin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Baowei; Lowry, David; Mayer, M. Uljana

2008-08-09

The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (ReAsH), which upon binding to an engineered tetracysteine binding motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the 1H- 15N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH.more » Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe (Kd = 0.36 ± 0.04 μM), which results in a reduction in the rate of ReAsH binding from 4900 M -1 sec -1 to 370 M -1 sec -1. In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe requires calcium-occupancy of amino-terminal sites (Kd = 18 ± 3 μM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.« less

The Structure of Rauvolfia serpentina Strictosidine Synthase Is a Novel Six-Bladed β-Propeller Fold in Plant Proteins[W

PubMed Central

Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

2006-01-01

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of ∼2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler–type reaction and represents a novel six-bladed β-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed β-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family. PMID:16531499

The structure of Rauvolfia serpentina strictosidine synthase is a novel six-bladed beta-propeller fold in plant proteins.

PubMed

Ma, Xueyan; Panjikar, Santosh; Koepke, Juergen; Loris, Elke; Stöckigt, Joachim

2006-04-01

The enzyme strictosidine synthase (STR1) from the Indian medicinal plant Rauvolfia serpentina is of primary importance for the biosynthetic pathway of the indole alkaloid ajmaline. Moreover, STR1 initiates all biosynthetic pathways leading to the entire monoterpenoid indole alkaloid family representing an enormous structural variety of approximately 2000 compounds in higher plants. The crystal structures of STR1 in complex with its natural substrates tryptamine and secologanin provide structural understanding of the observed substrate preference and identify residues lining the active site surface that contact the substrates. STR1 catalyzes a Pictet-Spengler-type reaction and represents a novel six-bladed beta-propeller fold in plant proteins. Structure-based sequence alignment revealed a common repetitive sequence motif (three hydrophobic residues are followed by a small residue and a hydrophilic residue), indicating a possible evolutionary relationship between STR1 and several sequence-unrelated six-bladed beta-propeller structures. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-309 in catalysis. The data will aid in deciphering the details of the reaction mechanism of STR1 as well as other members of this enzyme family.

Spatial and temporal distribution of the vibrionaceae in coastal waters of Hawaii, Australia, and France.

PubMed

Jones, B W; Maruyama, A; Ouverney, C C; Nishiguchi, M K

2007-08-01

Relatively little is known about large-scale spatial and temporal fluctuations in bacterioplankton, especially within the bacterial families. In general, however, a number of abiotic factors (namely, nutrients and temperature) appear to influence distribution. Community dynamics within the Vibrionaceae are of particular interest to biologists because this family contains a number of important pathogenic, commensal, and mutualist species. Of special interest to this study is the mutualism between sepiolid squids and Vibrio fischeri and Vibrio logei, where host squids seed surrounding waters daily with their bacterial partners. This study seeks to examine the spatial and temporal distribution of the Vibrionaceae with respect to V. fischeri and V. logei in Hawaii, southeastern Australia, and southern France sampling sites. In particular, we examine how the presence of sepiolid squid hosts influences community population structure within the Vibrionaceae. We found that abiotic (temperature) and biotic (host distribution) factors both influence population dynamics. In Hawaii, three sites within squid host habitat contained communities of Vibrionaceae with higher proportions of V. fischeri. In Australia, V. fischeri numbers at host collection sites were greater than other populations; however, there were no spatial or temporal patterns seen at other sample sites. In France, host presence did not appear to influence Vibrio communities, although sampled populations were significantly greater in the winter than summer sampling periods. Results of this study demonstrate the importance of understanding how both abiotic and biotic factors interact to influence bacterial community structure within the Vibrionaceae.

Angiotensin-I-converting enzyme and its relatives

PubMed Central

Riordan, James F

2003-01-01

Angiotensin-I-converting enzyme (ACE) is a monomeric, membrane-bound, zinc- and chloride-dependent peptidyl dipeptidase that catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a carboxy-terminal dipeptide. ACE has long been known to be a key part of the renin angiotensin system that regulates blood pressure, and ACE inhibitors are important for the treatment of hypertension. There are two forms of the enzyme in humans, the ubiquitous somatic ACE and the sperm-specific germinal ACE, both encoded by the same gene through transcription from alternative promoters. Somatic ACE has two tandem active sites with distinct catalytic properties, whereas germinal ACE, the function of which is largely unknown, has just a single active site. Recently, an ACE homolog, ACE2, has been identified in humans that differs from ACE in being a carboxypeptidase that preferentially removes carboxy-terminal hydrophobic or basic amino acids; it appears to be important in cardiac function. ACE homologs (also known as members of the M2 gluzincin family) have been found in a wide variety of species, even in those that neither have a cardiovascular system nor synthesize angiotensin. X-ray structures of a truncated, deglycosylated form of germinal ACE and a related enzyme from Drosophila have been reported, and these show that the active site is deep within a central cavity. Structure-based drug design targeting the individual active sites of somatic ACE may lead to a new generation of ACE inhibitors, with fewer side-effects than currently available inhibitors. PMID:12914653

Aquatic assemblages of the highly urbanized Santa Ana River Basin, California

USGS Publications Warehouse

Brown, Larry R.; Burton, Carmen; Belitz, Kenneth

2005-01-01

We assessed the structure of periphyton, benthic macroinvertebrate, and fish assemblages and their associations with environmental variables at 17 sites on streams of the highly urbanized Santa Ana River basin in Southern California. All assemblages exhibited strong differences between highly urbanized sites in the valley and the least-impacted sites at the transition between the valley and undeveloped mountains. Results within the urbanized area differed among taxa. Periphyton assemblages were dominated by diatoms (>75% of total taxa). Periphyton assemblages within the urbanized area were not associated with any of the measured environmental variables, suggesting that structure of urban periphyton assemblages might be highly dependent on colonization dynamics. The number of Ephemeroptera, Trichoptera, and Plecoptera (EPT) taxa included in macroinvertebrate assemblages ranged from 0 to 6 at urbanized sites. Benthic macroinvertebrate assemblages had significant correlations with several environmental variables within the urban area, suggesting that stream size and permanence were important determinants of distribution among the species able to survive conditions in urban streams. Only 4 of 16 fish species collected were native to the drainage. Fish assemblages of urbanized sites included two native species, arroyo chub Gila orcuttii and Santa Ana sucker Catostomus santaanae, at sites that were intermediate in coefficient of variation of bank-full width, depth, bed substrate, and water temperature. Alien species dominated urbanized sites with lesser or greater values for these variables. These results suggest that urban streams can be structured to enhance populations of native fishes. Continued study of urban streams in the Santa Ana River basin and elsewhere will contribute to the basic understanding of ecological principles and help preserve the maximum ecological value of streams in highly urbanized areas.

Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

PubMed Central

Sigala, Paul A.; Kraut, Daniel A.; Caaveiro, Jose M. M.; Pybus, Brandon; Ruben, Eliza A.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel

2009-01-01

Enzymes are classically proposed to accelerate reactions by binding substrates within active site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1 – 1 Å scale of most substrate rearrangements. The flexibility of active site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together high resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition state analog binding measurements to test the distance scale on which non-covalent forces can constrain side chain and ligand relaxation or translation along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function. PMID:18808119

In Silico and In Vitro Investigations of the Mutability of Disease-Causing Missense Mutation Sites in Spermine Synthase

PubMed Central

Zhang, Zhe; Norris, Joy; Schwartz, Charles; Alexov, Emil

2011-01-01

Background Spermine synthase (SMS) is a key enzyme controlling the concentration of spermidine and spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable. Methodology/Principal Findings To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement. Conclusions/Significance The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs can be expected to occur even at sites at which missense mutations were found to cause diseases. PMID:21647366

Urban population genetics of slum-dwelling rats (Rattus norvegicus) in Salvador, Brazil.

PubMed

Kajdacsi, Brittney; Costa, Federico; Hyseni, Chaz; Porter, Fleur; Brown, Julia; Rodrigues, Gorete; Farias, Helena; Reis, Mitermayer G; Childs, James E; Ko, Albert I; Caccone, Adalgisa

2013-10-01

Throughout the developing world, urban centres with sprawling slum settlements are rapidly expanding and invading previously forested ecosystems. Slum communities are characterized by untended refuse, open sewers and overgrown vegetation, which promote rodent infestation. Norway rats (Rattus norvegicus) are reservoirs for epidemic transmission of many zoonotic pathogens of public health importance. Understanding the population ecology of R. norvegicus is essential to formulate effective rodent control strategies, as this knowledge aids estimation of the temporal stability and spatial connectivity of populations. We screened for genetic variation, characterized the population genetic structure and evaluated the extent and patterns of gene flow in the urban landscape using 17 microsatellite loci in 146 rats from nine sites in the city of Salvador, Brazil. These sites were divided between three neighbourhoods within the city spaced an average of 2.7 km apart. Surprisingly, we detected very little relatedness among animals trapped at the same site and found high levels of genetic diversity, as well as structuring across small geographical distances. Most F(ST) comparisons among sites were statistically significant, including sites <400 m apart. Bayesian analyses grouped the samples in three genetic clusters, each associated with distinct sampling sites from different neighbourhoods or valleys within neighbourhoods. These data indicate the existence of complex genetic structure in R. norvegicus in Salvador, linked to the heterogeneous urban landscape. Future rodent control measures need to take into account the spatial and temporal linkage of rat populations in Salvador, as revealed by genetic data, to develop informed eradication strategies. © 2013 John Wiley & Sons Ltd.

A Manual for Evaluating School Facilities.

ERIC Educational Resources Information Center

Reida, G.W.

This survey manual evaluates the important points of functionality of school facilities in logical order. Instructions are given for the use of the manual, and separate sections present guidelines for evaluation of the following--(1) site, (2) building structure, (3) administrative spaces, (4) classrooms, (5) special rooms, (6) general service…

NMR and enzymology of modified DNA/protein interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kennedy, M.A.

1994-12-31

We have found distinct DNA structure and base dynamics precisely at the TpA cleavage site in the TTTAAA AHA III endonuclease restriction sequence. Hence, the unusual base stacking and mobility found in this sequence may be important to the mechanism of enzymatic cleavage of the phophodiester bond.

Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.

ERIC Educational Resources Information Center

Groves, John T.

1985-01-01

Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

Habitat selection is unaltered after severe insect infestation: Concerns for forest-dependent species

Treesearch

Claire A. Zugmeyer; John L. Koprowski

2009-01-01

Severe disturbance may alter or eliminate important habitat structure that helps preserve food caches of foodhoarding species. Recent recolonization of an insect-damaged forest by the endangered Mt. Graham red squirrel (Tamiasciurus hudsonicus grahamensis) provided an opportunity to examine habitat selection for midden (cache) sites following...

10 CFR 72.122 - Overall requirements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... accidents. (2)(i) Structures, systems, and components important to safety must be designed to withstand the..., and (B) Appropriate combinations of the effects of normal and accident conditions and the effects of... ability to return to a safe condition in the event of an accident. (e) Proximity of sites. An ISFSI or MRS...

10 CFR 72.122 - Overall requirements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... accidents. (2)(i) Structures, systems, and components important to safety must be designed to withstand the..., and (B) Appropriate combinations of the effects of normal and accident conditions and the effects of... ability to return to a safe condition in the event of an accident. (e) Proximity of sites. An ISFSI or MRS...

10 CFR 72.122 - Overall requirements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... accidents. (2)(i) Structures, systems, and components important to safety must be designed to withstand the..., and (B) Appropriate combinations of the effects of normal and accident conditions and the effects of... ability to return to a safe condition in the event of an accident. (e) Proximity of sites. An ISFSI or MRS...

D Model Generation from Uav: Historical Mosque (masjid LAMA Nilai)

NASA Astrophysics Data System (ADS)

Nasir, N. H. Mohd; Tahar, K. N.

2017-08-01

Preserving cultural heritage and historic sites is an important issue. These sites are subjected to erosion and vandalism, and, as long-lived artifacts, they have gone through many phases of construction, damage and repair. It is important to keep an accurate record of these sites using the 3-D model building technology as they currently are, so that preservationists can track changes, foresee structural problems, and allow a wider audience to "virtually" see and tour these sites. Due to the complexity of these sites, building 3-D models is time consuming and difficult, usually involving much manual effort. This study discusses new methods that can reduce the time to build a model using the Unmanned Aerial Vehicle method. This study aims to develop a 3D model of a historical mosque using UAV photogrammetry. In order to achieve this, the data acquisition set of Masjid Lama Nilai, Negeri Sembilan was captured by using an Unmanned Aerial Vehicle. In addition, accuracy assessment between the actual and measured values is made. Besides that, a comparison between the rendering 3D model and texturing 3D model is also carried out through this study.

Sintering-resistant Single-Site Nickel Catalyst Supported by Metal-Organic Framework

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Zhanyong; Schweitzer, Neil; League, Aaron

2016-02-17

Developing supported single-site catalysts is an important goal in heterogeneous catalysis, since the well-defined active sites afford opportunities for detailed mechanistic studies, thereby facilitating the design of improved catalysts. We present herein a method for installing Ni ions uniformly and precisely on the node of a Zr-based MOF, NU-1000, in high density and large quantity (denoted as Ni-AIM) using atomic layer deposition (ALD) in a metal–organic framework (MOF) (AIM). Ni-AIM is demonstrated to be an efficient gas-phase hydrogenation catalyst upon activation. The structure of the active sites in Ni-AIM is proposed, revealing its single-site nature. More importantly, due to themore » organic linker used to construct the MOF support, the Ni ions stay isolated throughout the hydrogenation catalysis, in accord with its long-term stability. A quantum chemical characterization of the catalyst and the catalytic process complements the experimental results. With validation of computational modeling protocols, we further targeted ethylene oligomerization catalysis by Ni-AIM guided by theoretical prediction. Given the generality of the AIM methodology, this emerging class of materials should prove ripe for the discovery of new catalysts for the transformation of volatile substrates.« less

Spatial variability in recruitment of benthos near drilling sites in the Iheya North hydrothermal field in the Okinawa Trough

NASA Astrophysics Data System (ADS)

Nakamura, Masako; Nakajima, Yuichi; Watanabe, Hiromi Kayama; Sasaki, Takenori; Yamamoto, Hiroyuki; Mitarai, Satoshi

2018-05-01

Due to increasing anthropogenic impacts on deep-sea hydrothermal vent ecosystems, it is essential to understand population structure and maintenance through larval recruitment and recovery of vent faunas after disturbances. In this study, we quantified vent animal recruitment in the Okinawa Trough, in the western Pacific Ocean. This is the first study to investigate recruitment patterns at a man-made hydrothermal vent. Colonization plates were deployed at three sites. Site 1 manifested new hydrothermal shimmering with small chimneys, white bacterial mats, and some alvinocaridid shrimp that arrived after drilling. Site 2 showed no evidence of newly arrived foundation species after drilling, and Site 3 had pre-existing animal communities in the vicinity of the new vent. Twenty-two months after deployment, colonization plates were retrieved and recruited animals were inventoried. Species composition and abundance differed among sites, but relatively high similarity in species composition was observed at Sites 1 and 3, though not at Site 2. Newly established communities on the plates at Sites 1 and 2 (no pre-existing fauna) showed lower species richness and abundance than at Site 3. Differences in abundance and size-frequency distributions of major recruits on the plates (i.e. Lepetodrilus nux, Bathymodiolus spp.) suggest the importance of reproductive and early life-history characteristics in spatial variability of recruitment. Lepetodrilus nux populations established on the plates at Site 1 showed high genetic connectivity. These results illustrate the importance of localized recruitment, which may have a significant impact on sustainability of vent faunal populations, despite the existence of regional metapopulations.

Structural basis for a hand-like site in the calcium sensor CatchER with fast kinetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Ying; Reddish, Florence; Tang, Shen

2013-12-01

High-resolution crystal structures of the designed calcium sensor CatchER revealed snapshots of calcium and gadolinium ions binding within the designed site in agreement with its fast kinetics. Calcium ions, which are important signaling molecules, can be detected in the endoplasmic reticulum by an engineered mutant of green fluorescent protein (GFP) designated CatchER with a fast off-rate. High resolution (1.78–1.20 Å) crystal structures were analyzed for CatchER in the apo form and in complexes with calcium or gadolinium to probe the binding site for metal ions. While CatchER exhibits a 1:1 binding stoichiometry in solution, two positions were observed for eachmore » of the metal ions bound within the hand-like site formed by the carboxylate side chains of the mutated residues S147E, S202D, Q204E, F223E and T225E that may be responsible for its fast kinetic properties. Comparison of the structures of CatchER, wild-type GFP and enhanced GFP confirmed that different conformations of Thr203 and Glu222 are associated with the two forms of Tyr66 of the chromophore which are responsible for the absorbance wavelengths of the different proteins. Calcium binding to CatchER may shift the equilibrium for conformational population of the Glu222 side chain and lead to further changes in its optical properties.« less

Effects of riparian forest removal on the trophic dynamics of a Neotropical stream fish assemblage.

PubMed

Lobón-Cerviá, J; Mazzoni, R; Rezende, C F

2016-07-01

The effects of riparian forest removal on a neotropical stream fish assemblage were assessed in the Mata Atlântica. Fish assemblage structure and fish feeding patterns were quantified at three sites along a pristine-to-deforested gradient in a Serra do Mar stream: (1) a pristine site fully covered by canopy with no light penetration and transparent waters, (2) an intermediate site with partially removed forest and (3) a fully removed forest site with no canopy and full light penetration where siltation and turbid waters predominate. Fish assemblage structure, fish densities and their feeding patterns differed widely among sites. Whilst the same five fish species occurred at the three sites, forest removal favoured the occurrence of sediment-tolerant iliophagous benthic species at the deforested site. At the pristine site, invertebrate prey predominated in water column fish diet and feeding overlap among species was low. Severe shifts in the feeding patterns were noticed in both deforested sites. Invertebrates were replaced by detritus, organic matter and algae at both sites and feeding overlap increased markedly. The overwhelming feeding adaptability of these neotropical fishes appeared capable of buffering the deleterious effects of forest removal on stream quality in terms of increased light penetration, siltation and water turbidity. Forest cutting in this Mata Atlântica stream clearly caused strong functional changes associated with forest clearance through important modifications in the assemblage organization and trophic patterns of the main species, but did not eliminate species. © 2016 The Fisheries Society of the British Isles.

Structural organization of intercellular channels II. Amino terminal domain of the connexins: sequence, functional roles, and structure.

PubMed

Beyer, Eric C; Lipkind, Gregory M; Kyle, John W; Berthoud, Viviana M

2012-08-01

The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics. Copyright © 2011. Published by Elsevier B.V.

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases.

PubMed

Cahn, Jackson K B; Brinkmann-Chen, Sabine; Spatzal, Thomas; Wiig, Jared A; Buller, Andrew R; Einsle, Oliver; Hu, Yilin; Ribbe, Markus W; Arnold, Frances H

2015-06-15

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site. © The Authors Journal Compilation © 2015 Biochemical Society.

Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures1[w

PubMed Central

Loke, Johnny C.; Stahlberg, Eric A.; Strenski, David G.; Haas, Brian J.; Wood, Paul Chris; Li, Qingshun Quinn

2005-01-01

Using a novel program, SignalSleuth, and a database containing authenticated polyadenylation [poly(A)] sites, we analyzed the composition of mRNA poly(A) signals in Arabidopsis (Arabidopsis thaliana), and reevaluated previously described cis-elements within the 3′-untranslated (UTR) regions, including near upstream elements and far upstream elements. As predicted, there are absences of high-consensus signal patterns. The AAUAAA signal topped the near upstream elements patterns and was found within the predicted location to only approximately 10% of 3′-UTRs. More importantly, we identified a new set, named cleavage elements, of poly(A) signals flanking both sides of the cleavage site. These cis-elements were not previously revealed by conventional mutagenesis and are contemplated as a cluster of signals for cleavage site recognition. Moreover, a single-nucleotide profile scan on the 3′-UTR regions unveiled a distinct arrangement of alternate stretches of U and A nucleotides, which led to a prediction of the formation of secondary structures. Using an RNA secondary structure prediction program, mFold, we identified three main types of secondary structures on the sequences analyzed. Surprisingly, these observed secondary structures were all interrupted in previously constructed mutations in these regions. These results will enable us to revise the current model of plant poly(A) signals and to develop tools to predict 3′-ends for gene annotation. PMID:15965016

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia.

PubMed

Moberg, Per; Nilsson, Stefan; Ståhl, Annelie; Eriksson, Anna-Carin; Glaser, Elzbieta; Mäler, Lena

2004-03-05

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.

Computational assessment of the cooperativity between RNA binding proteins and MicroRNAs in Transcript Decay.

PubMed

Jiang, Peng; Singh, Mona; Coller, Hilary A

2013-01-01

Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3'UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3'UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.

Geochemical analyses reveal the importance of environmental history for blue carbon sequestration

NASA Astrophysics Data System (ADS)

Kelleway, J. J.; Saintilan, N.; Macreadie, P. I.; Baldock, J. A.; Heijnis, H.; Zawadzki, A.; Gadd, P.; Jacobsen, G.; Ralph, P. J.

2017-07-01

Coastal habitats including saltmarshes and mangrove forests can accumulate and store significant blue carbon stocks, which may persist for millennia. Despite this implied stability, the distribution and structure of intertidal-supratidal wetlands are known to respond to changes imposed by geomorphic evolution, climatic, sea level, and anthropogenic influences. In this study, we reconstruct environmental histories and biogeochemical conditions in four wetlands of similar contemporary vegetation in SE Australia. The objective is to assess the importance of historic factors to contemporary organic carbon (C) stocks and accumulation rates. Results from the four cores—two collected from marine-influenced saltmarshes (Wapengo marine site (WAP-M) and Port Stephens marine site (POR-M)) and two from fluvial influenced saltmarshes (Wapengo fluvial site (WAP-F) and Port Stephens fluvial site (POR-F))—highlight different environmental histories and preservation conditions. High C stocks are associated with the presence of a mangrove phase below the contemporary saltmarsh sediments in the POR-M and POR-F cores. 13C nuclear magnetic resonance analyses show this historic mangrove root C to be remarkably stable in its molecular composition despite its age, consistent with its position in deep sediments. WAP-M and WAP-F cores did not contain mangrove root C; however, significant preservation of char C (up to 46% of C in some depths) in WAP-F reveals the importance of historic catchment processes to this site. Together, these results highlight the importance of integrating historic ecosystem and catchment factors into attempts to upscale C accounting to broader spatial scales.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

PubMed

Cioci, Gianluca; Mitchell, Edward P; Chazalet, Valerie; Debray, Henri; Oscarson, Stefan; Lahmann, Martina; Gautier, Catherine; Breton, Christelle; Perez, Serge; Imberty, Anne

2006-04-14

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

A mutational analysis and molecular dynamics simulation of quinolone resistance proteins QnrA1 and QnrC from Proteus mirabilis.

PubMed

Guo, Qinglan; Weng, Jingwei; Xu, Xiaogang; Wang, Minghua; Wang, Xiaoying; Ye, Xinyu; Wang, Wenning; Wang, Minggui

2010-10-08

The first report on the transferable, plasmid-mediated quinolone-resistance determinant qnrA1 was in 1998. Since then, qnr alleles have been discovered worldwide in clinical strains of Gram-negative bacilli. Qnr proteins confer quinolone resistance, and belong to the pentapeptide repeat protein (PRP) family. Several PRP crystal structures have been solved, but little is known about the functional significance of their structural arrangement. We conducted random and site-directed mutagenesis on qnrA1 and on qnrC, a newly identified quinolone-resistance gene from Proteus mirabilis. Many of the Qnr mutants lost their quinolone resistance function. The highly conserved hydrophobic Leu or Phe residues at the center of the pentapeptide repeats are known as i sites, and loss-of-function mutations included replacement of the i site hydrophobic residues with charged residues, replacing the i-2 site, N-terminal to the i residues, with bulky side-chain residues, introducing Pro into the β-helix coil, deletion of the N- and C-termini, and excision of a central coil. Molecular dynamics simulations and homology modeling demonstrated that QnrC overall adopts a stable β-helix fold and shares more similarities with MfpA than with other PRP structures. Based on homology modeling and molecular dynamics simulation, the dysfunctional point mutations introduced structural deformations into the quadrilateral β-helix structure of PRPs. Of the pentapeptides of QnrC, two-thirds adopted a type II β-turn, while the rest adopted type IV turns. A gap exists between coil 2 and coil 3 in the QnrC model structure, introducing a structural flexibility that is similar to that seen in MfpA. The hydrophobic core and the β-helix backbone conformation are important for maintaining the quinolone resistance property of Qnr proteins. QnrC may share structural similarity with MfpA.

PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality.

PubMed

Dehouck, Yves; Kwasigroch, Jean Marc; Gilis, Dimitri; Rooman, Marianne

2011-05-13

The rational design of modified proteins with controlled stability is of extreme importance in a whole range of applications, notably in the biotechnological and environmental areas, where proteins are used for their catalytic or other functional activities. Future breakthroughs in medical research may also be expected from an improved understanding of the effect of naturally occurring disease-causing mutations on the molecular level. PoPMuSiC-2.1 is a web server that predicts the thermodynamic stability changes caused by single site mutations in proteins, using a linear combination of statistical potentials whose coefficients depend on the solvent accessibility of the mutated residue. PoPMuSiC presents good prediction performances (correlation coefficient of 0.8 between predicted and measured stability changes, in cross validation, after exclusion of 10% outliers). It is moreover very fast, allowing the prediction of the stability changes resulting from all possible mutations in a medium size protein in less than a minute. This unique functionality is user-friendly implemented in PoPMuSiC and is particularly easy to exploit. Another new functionality of our server concerns the estimation of the optimality of each amino acid in the sequence, with respect to the stability of the structure. It may be used to detect structural weaknesses, i.e. clusters of non-optimal residues, which represent particularly interesting sites for introducing targeted mutations. This sequence optimality data is also expected to have significant implications in the prediction and the analysis of particular structural or functional protein regions. To illustrate the interest of this new functionality, we apply it to a dataset of known catalytic sites, and show that a much larger than average concentration of structural weaknesses is detected, quantifying how these sites have been optimized for function rather than stability. The freely available PoPMuSiC-2.1 web server is highly useful for identifying very rapidly a list of possibly relevant mutations with the desired stability properties, on which subsequent experimental studies can be focused. It can also be used to detect sequence regions corresponding to structural weaknesses, which could be functionally important or structurally delicate regions, with obvious applications in rational protein design.

Characteristics Associated with the Madden-Julian Oscillation at Manus Island

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Liping; McFarlane, Sally A.; Flaherty, Julia E.

2013-05-15

Ground-based high temporal and vertical resolution datasets from 2002 to 2008 of observations at the Atmospheric Radiation Measurement (ARM) Tropical Western Pacific (TWP) site on Manus Island, Papua New Guinea are used to examine the evolution of clouds and rainfall associated with the MJO life cycle. A composite MJO event is developed based on the NOAA MJO Index 4 using 13 events. The analysis shows that the cloud evolution during the composited MJO life cycle depicts a two-phase structure consisting of a development phase and a mature phase. During the development phase, congestus is the most important cloud type; duringmore » the mature phase, deep convection is the dominant cloud type. Consistent with this two-phase structure, the heavy rainfall frequency also shows a two-peak structure during the MJO life cycle. Light rainfall does not show a clear relation to the MJO life cycle, but shows variability on shorter time scales. From the development phase to the mature phase, the MJO structure shifts from the Type I to Type II structure, showing a different phase relationship between convection and dynamic fields (or wave motion) in the development and mature phases. During the shift, mid-level clouds play an important role in moving moisture to the mid-troposphere and preparing the atmosphere for the following deep convection. The discharge-recharge theory explains some of observed features of the MJO evolution at the ARM TWP Manus Island site.« less

Structure of MRDI Explains its Dual Function as a Metabolic Enzyme and a Mediator of Cell Invasion

PubMed Central

Templeton, Paul D.; Litman, Elizabeth S.; Metzner, Sandra I.; Ahn, Natalie G.; Sousa, Marcelo C.

2013-01-01

Metastatic melanoma is among the most intractable cancers to treat, where patients show resistance to therapy and limited survival time. A critical step in the development of metastatic melanoma is the acquisition of invasion and transition from thin to thick tumors on the skin, followed by invasion to lymph nodes. Prior studies have shown that metastatic melanoma is associated with dysregulation of RhoA and enhanced expression of a protein named “mediator of RhoA-dependent invasion (MRDI)”. Importantly, MRDI is a “moonlighting” enzyme, with two distinct functions in melanoma cells. First, MRDI acts as a methylthioribose-1-phosphate (MTR-1-P) isomerase, catalyzing a critical step in methionine salvage. Second, MRDI promotes and is necessary for melanoma cell invasion, independent of its catalytic activity. Here, we demonstrate that MtnA, a bacterial MTR-1-P isomerase, rescues the methionine salvage function of MRDI, but is unable to rescue its role in invasion. We then solve the crystal structure of MRDI to a resolution of 2.5 Å, in order to identify structural elements important for its invasion activity. We present this structure and its comparison with other MTR-1-P isomerases, and identify mutations within a region separate from the MTR-1-P binding site which interfere with invasion. Thus, structural elements in MRDI distal from the MTR-1-P catalytic site are responsible for the invasion phenotype. PMID:23859498

Using decision trees to understand structure in missing data

PubMed Central

Tierney, Nicholas J; Harden, Fiona A; Harden, Maurice J; Mengersen, Kerrie L

2015-01-01

Objectives Demonstrate the application of decision trees—classification and regression trees (CARTs), and their cousins, boosted regression trees (BRTs)—to understand structure in missing data. Setting Data taken from employees at 3 different industrial sites in Australia. Participants 7915 observations were included. Materials and methods The approach was evaluated using an occupational health data set comprising results of questionnaires, medical tests and environmental monitoring. Statistical methods included standard statistical tests and the ‘rpart’ and ‘gbm’ packages for CART and BRT analyses, respectively, from the statistical software ‘R’. A simulation study was conducted to explore the capability of decision tree models in describing data with missingness artificially introduced. Results CART and BRT models were effective in highlighting a missingness structure in the data, related to the type of data (medical or environmental), the site in which it was collected, the number of visits, and the presence of extreme values. The simulation study revealed that CART models were able to identify variables and values responsible for inducing missingness. There was greater variation in variable importance for unstructured as compared to structured missingness. Discussion Both CART and BRT models were effective in describing structural missingness in data. CART models may be preferred over BRT models for exploratory analysis of missing data, and selecting variables important for predicting missingness. BRT models can show how values of other variables influence missingness, which may prove useful for researchers. Conclusions Researchers are encouraged to use CART and BRT models to explore and understand missing data. PMID:26124509

QSAR and molecular graphics analysis of N2-phenylguanines as inhibitors of herpes simplex virus thymidine kinases.

PubMed

Gaudio, A C; Richards, W G; Takahata, Y

2000-02-01

A quantitative structure-activity relationship study of N2-(substituted)-phenylguanines (PHG) as inhibitors of herpes simplex virus thymidine kinase (HSV TK) was performed. The activity of a set of PHG derivatives were analyzed against the thymidine kinase of herpes simplex virus types 1 (HSV1 TK) and 2 (HSV2 TK). Classic and calculated physicochemical parameters were included in the analysis. The results showed that there is an important difference in the activity of the meta substituted PHG derivatives against HSV1 TK and HSV2 TK. The activity of the meta derivatives against HSV2 TK is influenced by a steric effect, which is not observed against HSV1 TK. The superposition of the three-dimensional structures of the active sites of HSV1 TK (crystal structure) and HSV2 TK (homology model) revealed that the amino acid Ile97 is located near the meta position in the HSV1 TK active site, whereas the amino acid Leu97 is located near the meta position in the HSV2 TK active site. This single difference in the active sites of both enzymes can explain the source of the steric effect and serves as an indication that our previously proposed binding mode for the PHG derivatives is plausible. However, another observed mutation in the active site region, Ala168 by Ser168, suggests that an alternative binding mode, similar to that of ganciclovir, could be possible.

Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

NASA Astrophysics Data System (ADS)

Shinde, Ranajit Nivrutti; Karthikeyan, Subramanian; Singh, Balvinder

2017-01-01

3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes the conversion of D-ribulose 5-phosphate (Ru5P) to L-3,4-dihydroxy-2-butanone-4-phosphate in the presence of Mg2+. Although crystal structures of DHBPS in complex with Ru5P and non-catalytic metal ions have been reported, structure with Ru5P along with Mg2+ is still elusive. Therefore, mechanistic role played by Mg2+ in the structure of DHBPS is poorly understood. In this study, molecular dynamics simulations of DHBPS-Ru5P complex along with Mg2+ have shown entry of Mg2+ from bulk solvent into active site. Presence of Mg2+ in active site has constrained conformations of Ru5P and has reduced flexibility of loop-2. Formation of hydrogen bonds among Thr-108 and residues - Gly-109, Val-110, Ser-111, and Asp-114 are found to be critical for entry of Mg2+ into active site. Subsequent in silico mutations of residues, Thr-108 and Asp-114 have substantiated the importance of these interactions. Loop-4 of one monomer is being proposed to act as a “lid” covering the active site of other monomer. Further, the conserved nature of residues taking part in the transfer of Mg2+ suggests the same mechanism being present in DHBPS of other microorganisms. Thus, this study provides insights into the functioning of DHBPS that can be used for the designing of inhibitors.

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2011-01-01

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems. PMID:21998754

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

PubMed

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Reconstruction of the inner structure of small scale mining waste dumps by combining GPR and ERTdata.

NASA Astrophysics Data System (ADS)

Kniess, Rudolf; Martin, Tina

2015-04-01

Two abandoned small waste dumps in the west of the Harz mountains (Germany) were analysed using ground penetrating radar (GPR) and electrical resistivity tomography (ERT). Aim of the project (ROBEHA, funded by the German Federal Ministry of Education and Research (033R105)) is the assessment of the recycling potential of the mining residues taking into account environmental risks of reworking the dump site. One task of the geophysical prospection is the investigation of the inner structure of the mining dump. This is important for the estimation of the approximate volume of potentially reusable mining deposits within the waste dump. The two investigated dump sites are different in age and therefore differ in their structure. The older residues (< 1930) consist of ore processing waste from density separation (stamp mill sand). The younger dump site descends from comprises slag dump waste. The layer of fine grained residues at the first dump site is less than 6 m thick and the slag layer is less than 2 m thick. Both sites are partially overlain by forest or grassland vegetation and characterized by topographical irregularities. Due to the inhomogeneity of the sites we applied electrical resistivity tomography (ERT) and ground penetrating radar (GPR) for detailed investigation. Using ERT we could distinguish various layers within the mining dumps. The resistivities of the dumped material differ from the bedrock resistivities at both sites. The GPR measurements show near surface layer boundaries down to 3 - 4 m. In consecutive campaigns 100 MHz and 200 MHz antennas were used. The GPR results (layer boundaries) were included into the ERT inversion algorithm to enable more precise and stable resistivity models. This needs some special preprocessing steps. The 3D-Position of every electrode from ERT measurement and the GPR antenna position on the surface require an accuracy of less than 1cm. At some points, the layer boundaries and radar wave velocities can be calibrated with borehole stratigraphic data from a mineralogical drilling campaign. This is important for a precise time-depth conversion of reflectors from GPR measurement. This reflectors were taken from radargram and have been adopted as resistivity boundary in the start model of the geoelectric inversion algorithm.

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

PubMed

Horowitz, Scott; Dirk, Lynnette M A; Yesselman, Joseph D; Nimtz, Jennifer S; Adhikari, Upendra; Mehl, Ryan A; Scheiner, Steve; Houtz, Robert L; Al-Hashimi, Hashim M; Trievel, Raymond C

2013-10-16

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase, Key Enzymes of Monolignol Biosynthesis[C][W

PubMed Central

Pan, Haiyun; Zhou, Rui; Louie, Gordon V.; Mühlemann, Joëlle K.; Bomati, Erin K.; Bowman, Marianne E.; Dudareva, Natalia; Dixon, Richard A.; Noel, Joseph P.; Wang, Xiaoqiang

2014-01-01

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates. PMID:25217505

A computational prediction of structure and function of novel homologue of Arabidopsis thaliana Vps51/Vps67 subunit in Corchorus olitorius.

PubMed

Zaman, Aubhishek; Fancy, Nurun Nahar

2012-12-01

Vps mediated vesicular transport is important for transferring macromolecules trapped inside a vesicle. Although highly abundant, Vps shows tremendous sequence variation among diverse array of species. However, this difference in sequence, which seems to also translate into substantial functional variation, is hardly characterized in Corchorus spp. Here, our computational study investigates structural and functional features of one of the Vps subunit namely Vps51/Vps67 in C. olitorius. Broad scale structural characterization revealed novel information about the overall Vps structure and binding sites. Moreover, functional analyses indicate interaction partners which were unexplored to date. Since membrane trafficking is essentially associated with nutrient uptake and chemical de-toxification, characterization of the Vps subunit can well provide us with better insight into important agronomic traits such as stress response, immune response and phytoremediation capacity.

Crystal Structure of Menin Reveals Binding Site for Mixed Lineage Leukemia (MLL) Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murai, Marcelo J.; Chruszcz, Maksymilian; Reddy, Gireesh

2014-10-02

Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostellamore » menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an {alpha}-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two {alpha}-helical bundles and covered by a {beta}-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.« less

Insights from molecular modeling and dynamics simulation of pathogen resistance (R) protein from brinjal.

PubMed

Shrivastava, Dipty; Nain, Vikrant; Sahi, Shakti; Verma, Anju; Sharma, Priyanka; Sharma, Prakash Chand; Kumar, Polumetla Ananda

2011-01-22

Resistance (R) protein recognizes molecular signature of pathogen infection and activates downstream hypersensitive response signalling in plants. R protein works as a molecular switch for pathogen defence signalling and represent one of the largest plant gene family. Hence, understanding molecular structure and function of R proteins has been of paramount importance for plant biologists. The present study is aimed at predicting structure of R proteins signalling domains (CC-NBS) by creating a homology model, refining and optimising the model by molecular dynamics simulation and comparing ADP and ATP binding. Based on sequence similarity with proteins of known structures, CC-NBS domains were initially modelled using CED- 4 (cell death abnormality protein) and APAF-1 (apoptotic protease activating factor) as multiple templates. The final CC-NBS structural model was built and optimized by molecular dynamic simulation for 5 nanoseconds (ns). Docking of ADP and ATP at active site shows that both ligand bind specifically with same residues and with minor difference (1 Kcal/mol) in binding energy. Sharing of binding site by ADP and ATP and low difference in their binding site makes CC-NBS suitable for working as molecular switch. Furthermore, structural superimposition elucidate that CC-NBS and CARD (caspase recruitment domains) domain of CED-4 have low RMSD value of 0.9 A° Availability of 3D structural model for both CC and NBS domains will . help in getting deeper insight in these pathogen defence genes.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Analysis of the Binding Sites of Porcine Sialoadhesin Receptor with PRRSV

PubMed Central

Jiang, Yibo; Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Kadariya, Ishwari; Cheng, Zhangrui; Ren, Yuwei; Chen, Xing; Zhou, Ao; Yang, Liguo; Kong, Dexin; Zhang, Shujun

2013-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) can infect pigs and cause enormous economic losses to the pig industry worldwide. Porcine sialoadhesin (pSN) and CD163 have been identified as key viral receptors on porcine alveolar macrophages (PAM), a main target cell infected by PRRSV. In this study, the protein structures of amino acids 1–119 from the pSN and cSN (cattle sialoadhesin) N-termini (excluding the 19-amino acid signal peptide) were modeled via homology modeling based on mSN (mouse sialoadhesin) template structures using bioinformatics tools. Subsequently, pSN and cSN homology structures were superposed onto the mSN protein structure to predict the binding sites of pSN. As a validation experiment, the SN N-terminus (including the wild-type and site-directed-mutant-types of pSN and cSN) was cloned and expressed as a SN-GFP chimera protein. The binding activity between SN and PRRSV was confirmed by WB (Western blotting), FAR-WB (far Western blotting), ELISA (enzyme-linked immunosorbent assay) and immunofluorescence assay. We found that the S107 amino acid residue in the pSN N-terminal played a crucial role in forming a special cavity, as well as a hydrogen bond for enhancing PRRSV binding during PRRSV infection. S107 may be glycosylated during PRRSV infection and may also be involved in forming the cavity for binding PRRSV along with other sites, including W2, Y44, S45, R97, R105, W106 and V109. Additionally, S107 might also be important for pSN binding with PRRSV. However, the function of these binding sites must be confirmed by further studies. PMID:24351868

Modeling marbled murrelet (Brachyramphus marmoratus) habitat using LiDAR-derived canopy data

USGS Publications Warehouse

Hagar, Joan C.; Eskelson, Bianca N.I.; Haggerty, Patricia K.; Nelson, S. Kim; Vesely, David G.

2014-01-01

LiDAR (Light Detection And Ranging) is an emerging remote-sensing tool that can provide fine-scale data describing vertical complexity of vegetation relevant to species that are responsive to forest structure. We used LiDAR data to estimate occupancy probability for the federally threatened marbled murrelet (Brachyramphus marmoratus) in the Oregon Coast Range of the United States. Our goal was to address the need identified in the Recovery Plan for a more accurate estimate of the availability of nesting habitat by developing occupancy maps based on refined measures of nest-strand structure. We used murrelet occupancy data collected by the Bureau of Land Management Coos Bay District, and canopy metrics calculated from discrete return airborne LiDAR data, to fit a logistic regression model predicting the probability of occupancy. Our final model for stand-level occupancy included distance to coast, and 5 LiDAR-derived variables describing canopy structure. With an area under the curve value (AUC) of 0.74, this model had acceptable discrimination and fair agreement (Cohen's κ = 0.24), especially considering that all sites in our sample were regarded by managers as potential habitat. The LiDAR model provided better discrimination between occupied and unoccupied sites than did a model using variables derived from Gradient Nearest Neighbor maps that were previously reported as important predictors of murrelet occupancy (AUC = 0.64, κ = 0.12). We also evaluated LiDAR metrics at 11 known murrelet nest sites. Two LiDAR-derived variables accurately discriminated nest sites from random sites (average AUC = 0.91). LiDAR provided a means of quantifying 3-dimensional canopy structure with variables that are ecologically relevant to murrelet nesting habitat, and have not been as accurately quantified by other mensuration methods.

Evolutionary Origin and Conserved Structural Building Blocks of Riboswitches and Ribosomal RNAs: Riboswitches as Probable Target Sites for Aminoglycosides Interaction.

PubMed

Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl; Hejazi, Mohammad Saeid

2014-01-01

Riboswitches, as noncoding RNA sequences, control gene expression through direct ligand binding. Sporadic reports on the structural relation of riboswitches with ribosomal RNAs (rRNA), raises an interest in possible similarity between riboswitches and rRNAs evolutionary origins. Since aminoglycoside antibiotics affect microbial cells through binding to functional sites of the bacterial rRNA, finding any conformational and functional relation between riboswitches/rRNAs is utmost important in both of medicinal and basic research. Analysis of the riboswitches structures were carried out using bioinformatics and computational tools. The possible functional similarity of riboswitches with rRNAs was evaluated based on the affinity of paromomycin antibiotic (targeting "A site" of 16S rRNA) to riboswitches via docking method. There was high structural similarity between riboswitches and rRNAs, but not any particular sequence based similarity between them was found. The building blocks including "hairpin loop containing UUU", "peptidyl transferase center conserved hairpin A loop"," helix 45" and "S2 (G8) hairpin" as high identical rRNA motifs were detected in all kinds of riboswitches. Surprisingly, binding energies of paromomycin with different riboswitches are considerably better than the binding energy of paromomycin with "16S rRNA A site". Therefore the high affinity of paromomycin to bind riboswitches in comparison with rRNA "A site" suggests a new insight about riboswitches as possible targets for aminoglycoside antibiotics. These findings are considered as a possible supporting evidence for evolutionary origin of riboswitches/rRNAs and also their role in the exertion of antibiotics effects to design new drugs based on the concomitant effects via rRNA/riboswitches.

Characterizing the structural ensemble of γ-secretase using a multiscale molecular dynamics approach† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00980a Click here for additional data file.

PubMed Central

Aguayo-Ortiz, Rodrigo; Chávez-García, Cecilia; Straub, John E.

2017-01-01

γ-Secretase is an intramembrane-cleaving aspartyl protease that plays an essential role in the processing of a variety of integral membrane proteins. Its role in the ultimate cleavage step in the processing of amyloid precursor protein to form amyloid-β (Aβ) peptide makes it an important therapeutic target in Alzheimer's disease research. Significant recent advances have been made in structural studies of this critical membrane protein complex. However, details of the mechanism of activation of the enzyme complex remain unclear. Using a multiscale computational modeling approach, combining multiple coarse-grained microsecond dynamic trajectories with all-atom models, the structure and two conformational states of the γ-secretase complex were evaluated. The transition between enzymatic state 1 and state 2 is shown to critically depend on the protonation states of the key catalytic residues Asp257 and Asp385 in the active site domain. The active site formation, related to our γ-secretase state 2, is observed to involve a concerted movement of four transmembrane helices from the catalytic subunit, resulting in the required localization of the catalytic residues. Global analysis of the structural ensemble of the enzyme complex was used to identify collective fluctuations important to the mechanism of substrate recognition and demonstrate that the corresponding fluctuations observed were uncorrelated with structural changes associated with enzyme activation. Overall, this computational study provides essential insight into the role of structure and dynamics in the activation and function of γ-secretase. PMID:28970936

Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2-dehydrogenase

PubMed Central

Lucas, James E; Siegel, Justin B

2015-01-01

Enzyme active site residues are often highly conserved, indicating a significant role in function. In this study we quantitate the functional contribution for all conserved molecular interactions occurring within a Michaelis complex for mannitol 2-dehydrogenase derived from Pseudomonas fluorescens (pfMDH). Through systematic mutagenesis of active site residues, we reveal that the molecular interactions in pfMDH mediated by highly conserved residues not directly involved in reaction chemistry can be as important to catalysis as those directly involved in the reaction chemistry. This quantitative analysis of the molecular interactions within the pfMDH active site provides direct insight into the functional role of each molecular interaction, several of which were unexpected based on canonical sequence conservation and structural analyses. PMID:25752240

RF-Phos: A Novel General Phosphorylation Site Prediction Tool Based on Random Forest.

PubMed

Ismail, Hamid D; Jones, Ahoi; Kim, Jung H; Newman, Robert H; Kc, Dukka B

2016-01-01

Protein phosphorylation is one of the most widespread regulatory mechanisms in eukaryotes. Over the past decade, phosphorylation site prediction has emerged as an important problem in the field of bioinformatics. Here, we report a new method, termed Random Forest-based Phosphosite predictor 2.0 (RF-Phos 2.0), to predict phosphorylation sites given only the primary amino acid sequence of a protein as input. RF-Phos 2.0, which uses random forest with sequence and structural features, is able to identify putative sites of phosphorylation across many protein families. In side-by-side comparisons based on 10-fold cross validation and an independent dataset, RF-Phos 2.0 compares favorably to other popular mammalian phosphosite prediction methods, such as PhosphoSVM, GPS2.1, and Musite.